Their duty is to protect the Tree of Life and all who live there and who seek refuge there from those who might cause them harm. Unlike The Lion Guard, the Night Pride sleeps during the day, and acts during the night unless a situation comes up during daylight hours. This behaviour is true to real life lions because they are nocturnal animals and do all the hunting in the night time, while sleeping all day. This schedule can be exploited by anyone seeking to do harm to the Tree of Life by attacking at sunrise when the Night Pride is finishing their patrol.
When Bunga accidentally causes a rockslide blocking the main entrance to the Tree of Life, Rani, leader of the Night Pride, thinks that the Lion Guard is a threat. Her younger brother Baliyo goes up to see who the strangers are. He felt that the Lion Guard had hostile intentions due to Kion saying "nothing will stop them from getting to the Tree of Life" and attacks Kion so that the rest of the Night Pride are forced to attack the guard. Kion soon uses the Roar of the Elders on Baliyo, which sends him flying through the pass and Rani gets upset. Rani pounces on Kion and tells him that he and his friends will never be welcome at the Tree of Life. Baliyo meets up with the Night Pride again and Rani asks her brother if he's okay and he says that he's fine despite feeling dizzy. Rani decides to speak with her grandmother Janna. Rani tells Janna that the Night Pride fought off intruders which Janna praises her for, but then Rani mentions a lion having a powerful roar and Janna says that the roar has returned to the Tree of Life. Rani then asks about the scar on Kion's eye. Janna explains and tells her to remember "Sisi ni sawa". Janna tells her granddaughter to welcome the Lion Guard to the Tree of Life. After speaking to her Night Pride they all think that the Guard shouldn't come in. Rani soon talks to her late parents Sãhasí and Ãnanda to get advice. They say to listen to Janna and welcome the Guard into the Tree of Life. After finding the Guard, they notice that they cleared the path but remained outside. Kion apologizes for the rock slide and says that he and Ono need to be healed. Rani soon welcomes them to the Tree of Life.
Rani brings the Lion Guard and Makini inside the Tree of Life to speak with her grandmother, Queen Janna. She asks Nirmala to create a full diagnosis for Kion's and Ono's conditions. While Kion is at his first healing session, Janna asks Rani to show the rest of the Guard around the Tree of Life. Rani tells them all about the mystical place and says that everyone is welcome at the Tree of Life. Ullu then comes to tell Rani some important news and she leaves the Guard behind. She soon meets Makucha, Chuluun, and Ora. The Night Pride fights them off and the Lion Guard comes to help. However, Rani didn't want their help and asks them to leave as soon as possible. Then, Rani talks to her grandmother about this problem. She claims that she should have never let them in. Janna tells Kion to not worry about protecting the Tree of Life and to continue his healing. When Rani asks Nirmala if he is healed yet, she says that she is just like him, impatient. Makucha and the others come back again and the Night Pride fights them. With some help from the Lion Guard, they're able to win the battle.
Rani is hanging out with the Lion Guard and her brother. Soon Kion and Nirmala come along and see that Kion has mud on his face Nirmala says it's part of his healing. Nirmala says that he has a long way to go before he gets healed. She also says that it's time for Ono to get healed. Ono is excited and heads off to the Tree of Life. Fuli asks if Bunga can be any noisier when he eats. Baliyo says that Bunga should meet his friend Binga. Rani and her Night Pride soon see Mama Binturong being chased by Makucha's army. They save Mama and welcomes her to the Tree of Life. Kion asks if Rani is here for Ono's healing. She says that she's just letting Mama Binturong into the Tree of Life. After Mama Binturong finds out what happens she comes up with a plan. Rani takes her to a nice place to get some rest. The Night Pride hears that Makucha's army is attacking again. However, they spot Mama Binturong who says that she was attacked by Binga and Bunga. Bunga explains what happened and she runs off. Before she escapes too far Bunga and Binga stop her with their stink. Rani tells Ono that he was the one who figured out Mama Binturong's plan. She thanks Ono for his help Kion says that she's right. Ono is still getting used to his new role in the Guard.
Rani and Baliyo hear that there's a bad guy named "Poa the Destroyer" on the loose. After talking to the penguin, Pinguino, turns out that it's just Kion's friend Beshte. She asks Baliyo to round up the rest of the Lion Guard. Rani asks Kion how well he knows Beshte. He becomes scared as Rani tells Kion to come to the Tree of Life. Beshte wonders why the animals are scared of him. Rani explains and Beshte thinks he is a destroyer. Ono says once the animals get to know Beshte they're sure to like him. Rani tells the animals that there has been a misunderstanding. The Lion Guard tells the animals that Beshte isn't a bad guy. When the penguins show up Rani sees that Pinguino is missing. She says that he needs to be here since he caused the biggest fuss. Kion asks Anga to find Beshte and Pinguino. They soon show up and Pinguino says that Beshte is now Poa the Life Saver. The animals soon cheer for Beshte.
The Night Pride and the Lion Guard are stopping Makcuha's Army from preventing a family of Tigers from entering the Tree of Life. Makucha's Army is forced to retreat and Rani invites the tigers into the Tree of Life. Ullu soon comes by saying that Queen Janna would like to see her and the Night Pride along with Kion. Queen Janna, at the time, was incredibly weak and on the verge of death. Janna tells Rani that it's time for her to become queen but thinks she isn't ready and Queen Janna says she is. Rani starts to cry as Queen Janna passes away. Kion gives her his condolences and her thanks to him while crying on his shoulder as he sheds a tear as well. Makini soon tells the animals of the Tree of Life the sad news. At the funeral, Rani is sad and alone so Kion goes to talk to her. Rani talks about her admiration for her grandmother and is worried she won't measure up to her. Kion encourages her and opens up about his doubts when he started leading the Lion Guard. Kion tells her that he hasn't been talking to his grandfather Mufasa since he got his scar out of worry that he'll resemble Scar to Mufasa. Rani says we all have scars while showing him the scar on her forepaw while saying "si si nisawa" which is what her Grandmother said that convinced Rani to let Kion into the Tree of Life which is a decision Rani is now glad about. Rani tells Kion that just talking to Mufasa might make him feel better and Kion thanks her for the advice while telling Rani she's a lot like her Grandmother which leaves Rani taken aback by the comparison. Makini soon shows Rani all the paintings that she made in the Tree of Life. The tigers soon come back and ask if they can stay at the Tree of Life. Rani says they're welcomed and goes with Bunga to find them a new home. Rani goes to her coronation at sunset. Fuli, Anga, and Nirmala greet Rani with a song as she becomes queen and all the animals cheer for their new ruler. That night Rani tells her ghostly grandmother that she's ready to be queen.
Rani talks to Kion during his latest part in his healing treatment. Makucha's army comes back to attack the animals at the Tree of Life. Kion, by working together, the Night Pride and the Lion Guard stops them.
Kion decides that it's time for the Lion Guard to defend the Tree of Life during the day. Rani and the Night Pride goes to get some rest. Bunga goes to the Tree of Life to speak to Rani. Bunga wonders why Kion isn't ready to use his roar. Makini says that Kion is still working on his roar. Makini teaches Rani all about Askari the first leader of the Lion Guard. She says that Askari would go to Chikha Escarpment to practice the Roar. Rani and Bunga go to see Kion. They watch Kion uses the roar. Soon they hear about Makucha's Army attacking animals at the Tree of Life. Anga brings them and Kion uses his roar on them. He blasts the bad guys away and the animals are safe. After realizing the full power of Kion's roar, Rani asks now healed Kion to stay instead of return to the Pride Lands.
After speaking to Mufasa, Rani comes up to Kion. Rani asks Kion if he's made his choice on staying at the Tree of Life. They both think that they make a great team and they're of the same pride. As the Guard is ready to leave, Rani wishes that they'll stay. Everyone but Fuli wanted to stay too. Soon Azaad comes and Fuli asks how Rani knows him. She says that he's been to the Tree of Life before. She asks why he's here and he says that he was showing travelers how to get to the Tree of Life. Two of the Lion Guard's friends Janja and Jasiri come and tells the Guard that Zira and her Pride have come back into the Outlands. Kion knows that he must protect the Pride Lands from Zira. The Guard soon says bye to Rani and Rani hopes that the Roar will return to the Tree of Life someday.
After Kion relinquishes his role as the Leader of the Lion Guard to Vitani, Askari appears in the sky and tells Kion that there's another place that could use his help.
Kion realizes he's talking about the Tree of Life and the Mark of the Night Pride given to Kion by Queen Janna before her death appears on Kion's shoulder. Kion asks the old Guard if they would like to protect the Tree of Life and they all accept with the Mark of the Night Pride replacing the Mark of the Lion Guard on their bodies. Kion along with all of his family and friends arrives at the Tree of Life and watches Kion become Rani's mate and King of the Tree of Life.