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In 1526, Shao Jun returns to China after her journey to Europe, where she met the Italian Assassins' renowned Mentor Ezio Auditore da Firenze and was given a Precursor box to guide her on her quest. Shao Jun's mission is to eliminate the Eight Tigers, a group of Templar eunuchs who influenced the Jiajing Emperor into launching a purge of the Chinese Brotherhood. Shao Jun easily assassinates her first target, Gao Feng, in the Maijishan Grottoes, though she loses the Precursor box in the process. After rescuing her old teacher Hong Liwei, who was imprisoned by the Templars, Shao Jun escapes and regroups with her Mentor, Wang Yangming. Yangming reveals that he also killed a Tiger, Ma Yongcheng, leaving only four targets to eliminate: Gu Dayong, Wei Bin, Qiu Ju, and the Tigers' leader, Zhang Yong.

Later, Yangming is informed by a contact that Gu Dayong, who controls the slave trade in Macau, has the Precursor box. Shao Jun infiltrates the city and rescues a young boy named Xiao Hu, who reveals that his father is also an Assassin. Unable to save Xiao Hu's father, Shao Jun exacts revenge on Dayong, but learns that the Precuror box is no longer in Macau. In retaliation for Dayong's murder, Qiu Ju orders the guards to burn Macau's port, to show the Assassins the consequences of their actions. Shao Jun manages to save several civilians and escape, but the incident leaves her traumatized.

By 1529, Shao Jun is still struggling with her trauma and is uncertain if she is fighting the Templars simply out of revenge or to protect the people's freedom. Regardless, when Yangming arrives with news that Wei Bin is in Nan'an, Shao Jun decides to accompany her Mentor to the city. In the process, she is reunited with Xiao Hu, who survived the Macau fires and reveals that the people of the city consider her a hero. After Xiao Hu explains that the Templars are solely responsible for the atrocities they commit and that Shao Jun should not blame herself for other people's actions, the Assassin regains her resolve. She infiltrates Wei Bin's stronghold and kills him, only to learn that the other Tigers have lured Yangming into a trap. Arriving too late to save her Mentor, Shao Jun meets a Buddhist monk, who helps her organize Yangming's funeral and teaches her about the Precursors and the special abilities she inherited from them.

In 1530, Shao Jun infiltrates the Forbidden City to rescue her childhood friend, Empress Zhang Qijie. However, while meeting with Qijie, Shao Jun is confronted by Qiu Ju, who claims that the Empress has betrayed and lured her into a trap. Knowing her friend was forced to help the Templars, Shao Jun forgives her and fights Qiu Ju, who is killed by falling debris after the chamber they are in catches fire. Shao Jun and Qijie escape to safety and reconcile after the Assassin admits that she misjudged her friend, thinking Qijie needed her protection when she was more than capable of looking after herself.

By 1532, Shao Jun has begun training Xiao Hu as her Assassin apprentice. After learning that Zhang Yong is planning to let the Mongols led by Altan Khan invade China so he can maintain his grip on power, the two Assassins travel to the Great Wall to prevent the invasion. Shao Jun confronts Zhang Yong, who boasts that the Precursor box has been shipped out of China and that his death will not stop the Templars' plans. Shao Jun acknowledges that the Assassin-Templar war will continue for centuries to come, but declares that her conflict with Zhang Yong is over, and kills the Tiger leader.

Years later, an older Shao Jun and an adult Xiao Hu meet atop the Great Wall. Having completed his training, Xiao Hu retakes his birth name of Kotetsu and informs Shao Jun that he will be returning to his homeland of Japan to fight the Templars' growing influence and establish a new Assassin Brotherhood. Shao Jun wishes her apprentice good luck in his mission, and the two hug each other and recite the tenets of the Assassins' Creed before parting ways.