Ishii is an American of Chinese and Japanese heritage. When she was still very young, her parents were brutally murdered by a Yakuza crime lord, Boss Matsumoto. She then spent the remainder of her childhood years training herself to be a ruthless killing machine so that she could get revenge; she finally got it by posing as a child prostitute and stabbing him to death when she was eleven years old. By age twenty she was already one of the world's top assassins.

She came to be in the employ of Bill (ironically the very man who had killed her father and burned her home as one of Matsumoto's minions) and became a member of The Deadly Viper Assassination Squad, along with Beatrix Kiddo; her codename was Cottonmouth. She participated in the attack on Kiddo's wedding rehearsal, leaving Kiddo in a coma.

One year later, Ishii became the head of the Tokyo Yakuza, over the objections of the son of one of its founding members, Boss Tanaka, who looked down upon her mixed ethnic makeup. She stifled this criticism by decapitating him instantly. However, despite her ruthlessness, she told her subordinate bosses that they were free to question her on any of her decisions, and that she would take the time to convince them in a civilized manner. She also stated that no subject was to be taboo around her...except the one voiced by Boss Tanaka. If any of them were to bring up the subject of her heritage again, she would "collect their fucking head."

Ishii also had her own personal army of sword-wielding, masked punks known as the Crazy 88. She ruled over her criminal empire with the help of her lawyer, second lieutenant, and best friend, Sofie Fatale.

Ishii would become the first target of Beatrix Kiddo, since she was the easiest to find. In a Japanese nightclub called The House of Blue Leaves, Kiddo overcame not only the Crazy 88, but also Ishii's bodyguard, Gogo Yubari (Chiaki Kuriyama), who proved a challenge with her Meteor hammer weapon.

Ishii was eventually killed in a duel with Kiddo, who sliced off the crown of her head. The battle took place outside in a moonlit, snow-covered garden. This scene is a tribute to the cult film Lady Snowblood, on which O-Ren's character was based.