The Night Pride struggles to defend the Tree of Life from the Lion Guard, with Baliyo being blown away by Kion's Roar of the Elders. They back off after Rani orders the Guard to stay away from the Tree. After counseling with Janna, Rani informs the rest of the Night Pride that they are to invite the Guard to the Tree of Life after all. The pride returns and escorts the Lion Guard to the Tree.
The Night Pride leads the Lion Guard to their queen, Janna, who welcomes them for healing. Rani then gives the team a tour of the kingdom, while Nirmala leads Kion to his first task in his healing journey.
Later, when Makucha and his allies, Chuluun and Ora, attempt to invade the Tree of Life, the Night Pride blocks their entry. The team defeats them easily by working together. Afterward, the Lion Guard arrives too late to help.
The Night Pride fends off another attack by Makucha's army, this time by night. However, their third attack the following morning proves more difficult, as Ora bites and paralyzes Baliyo. Just in time, Kion arrives to help.
Mama Binturong plots to invade the Tree of Life by pretending to seek sanctuary from Makucha and his army. The army enacts the plan, and the Night Pride falls for it, accepting Mama Binturong for healing at the Tree of Life.
Mama Binturong returns to her allies and instructs them to kill Janna while she distracts the Night Pride. The plan goes into motion, with the Night Pride falling for Mama Binturong's cries for help. In the meantime, Makucha, Chuluun, and Ora sneak over the border.
After luring the Night Pride with cries for help, Mama Binturong attacks Bunga and Binga. The Night Pride arrives, and Mama Binturong attempts to blame the honey badgers for the fight. However, Ullu arrives and informs the Night Pride of Mama Binturong's deception. Bunga and Binga then use their stench to defeat Mama Binturong.
When the Night Pride arrives at the Tree of Life, they notice a crowd of animals gathering and realize that Janna is dying. Once inside the Tree of Life, Janna tells the pride to help Rani during her reign. Janna dies peacefully, and the pride grieves. They later attend Janna's funeral.
At Rani's coronation, the Night Pride cheers their new queen on.
One day, Makucha and his army launch an attack on the Tree of Life. The Night Pride challenges them, though Baliyo's yawning exposes how exhausted they are from a night of patrolling. The Lion Guard arrives in time to help, but Kion and Rani's dual leadership results in confusion.
Eventually, the Lion Guard and the Night Pride succeed in driving off the army. Kion offers to patrol the Tree of Life during the day in order to give the Night Pride time to rest, and Rani gratefully accepts his offer. Unbeknownst to the team, Mama Binturong is spying on their conversation from a nearby tree.
Later, Ullu warns the Lion Guard and the Night Pride that Makucha's army is launching another attack. The Night Pride arrives, and Kion leads the two teams in their attack.
After the fight, Bunga compliments Kion for his good leadership skills, and Nirmala agrees, adding that Kion's healing is complete. Rani worries that this will prompt Kion to return to the Pride Lands, but Kion assures her that he and the Lion Guard will help defend the Tree of Life from Makucha's army first.
Later, Mama Binturong brings in new allies for Makucha's army, including Fahari, Jiona, two komodo dragons, and Smun and his prickle. Makucha's army invades the Tree of Life. Ullu warns the Night Pride about the army's attack, and they rush to the Tree of Life, only to be shut in by a giant rock pushed by Ora and his minions. With the Night Pride out of commission, the army attacks the animals at the Tree of Life.
The Lion Guard arrives, having been warned by Ullu, and resolves to free the Night Pride from the army's trap. Beshte attempts to move the rock from the Tree of Life's entrance, but is thwarted by Ora and his minions. Just then, Kion, Bunga, and Rani arrive, and Kion uses the Roar to defeat the villains. He then strikes the rock at the Tree of Life's entrance with lightning, destroying it and freeing the Night Pride. Finally, Kion sweeps Makucha and his army into a tornado, which sends them far away from the Tree of Life to never be seen again.
Kion seeks advice from the spirit of his grandfather, Mufasa, about whether he should return to the Pride Lands or not. He explains that he could use more training with the Roar of the Elders and that the Night Pride needs help protecting the Tree of Life. Mufasa tells Kion to choose the path that is best for him.
Later, Rani asks Kion if he will be her king. However, before he can answer, the Lion Guard approaches and declares that they are ready to return home. Ullu approaches, heralding the arrival of Azaad, Janja, and Jasiri. Jasiri informs Kion that Zira is invading the Pride Lands and that the royal family needs the Lion Guard's help. While Kion decides to return home to help, Makini stays behind due to her duty as Rani's Royal Mjuzi.