The Tree of Life is a location that is home to Rani, Kion, the Night Pride, and many rare animals. It is the true home and origin of the Roar of the Elders. In Season 3 of The Lion Guard, said group along with Makini travel there to heal Kion and Ono's wounds.
The Tree of Life is a safe-haven where the animals from everywhere come to heal or if they are of an endangered species. Rafiki told the Lion Guard about it near the end of Battle for the Pride Lands. They hope to receive help for problems for which they can't find any solutions elsewhere. The Tree of Life has all kinds of animal habitats.
The Tree of Life appears to have mystical powers, as Queen Janna channeled its energy to help heal Ono's eyes to restore his vision. Though this power appears to have limits, as Janna could not restore Ono's vision to be as keen as it used to be. However, after Janna's death in the episode Long Live the Queen, Rani takes over as the new queen of the Tree of Life.
After Kion and Ono get workplace injuries during the final battle against Scar, Rafiki advises that they and the rest of the Lion Guard set off to find the Tree of Life to find the cures for their conditions. It is vital for Kion to find this tree, because it's the only place he can find a treatment for the effects of Ushari's venom, which got into his left eye when he got his own scar.
The Lion Guard has finally completed their journey to the Tree of Life but are confronted by the Night Pride; a different pride of lions who safeguard the tree and its inhabitants. When Kion's roar accidentally causes an avalanche, Ullu, an owl who guards the entrance, alerts the Night Pride thinking the Lion Guard is trying to attack. At first, Rani, leader of the Night Pride, is hesitant to let them in. However, she soon agrees too. Once the Lion Guard, has entered the valley they gaze upon the Tree of Life in person for the first time.
Rani brings the Lion Guard inside the Tree of Life to speak with her grandmother Janna. Janna asks Nirmala to help heal Kion's scar. While Kion is getting healed by Janna, she asks Rani to show the rest of the guard around the Tree of Life. Rani tells them all about the Tree of Life and says that everyone is accepted to be welcomed the tree. Ullu comes to tell Rani some important news. Rani leaves the guard behind. They soon meet Makucha, Chuluun and Ora. The Night Pride fights them off, but Kion and the Lion Guard arrive after it's all over and crash into her. Rani didn't want their help and asks them to leave as soon as possible. After hearing the descriptions of the animals the Night Pride fought, Kion realizes that Makucha and the others must have followed them. This greatly angers Rani as it confirmed her worst fears about something bad happening if she let the Lion Guard into the Tree of Life as there is now a group of vicious predators on her doorstep. Rani talks to her grandmother about this problem and claims that they should have never let Kion and the Lion Guard in. Janna tells Kion to not worry about protecting the Tree of Life and to continue his healing. Rani asks Nirmala if Kion is healed yet. She says that she is just like him. Makucha and the others come back again and the Night Pride fights them. Baliyo gets bitten by Ora's venom and is left incapacitated. Kion completes his task of getting the flower and helps fight off Makucha and Chuluun. Rani then thanks the Lion Guard for their help.