Gallic acid (GA), a natural polyphenol, has been shown to exert a variety of heath promoting effects. We herein investigated the critical role of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response in the protection of GA against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in L02 cells. Pretreatment of GA prevented the hepatocytotoxicity induced by t-BHP, as evidenced by the facts that GA suppressed t-BHP-induced cytotoxicity and reactive oxygen species (ROS) generation. GA induced nuclear translocation of Nrf2 along with expression of target proteins, including heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic modify subunit (GCLC), and increased intracellular glutathione (GSH) content. Additionally, GA induced phosphorylated activation of extracellular regulated kinase (ERK), and ERK inhibitor PD98059 partially decreased GA-induced hepatoprotection, and downregulated the increased protein expressions of Nrf2, GCLC and HO-1 induced by GA. Interestingly, we found that GA could enhance the thermal stability of Keap1, which indicated the potential interaction between GA and Keap1. Furthermore, molecular docking indicated that GA possibly competed with Nrf2 for binding to Keap1. Collectively, GA effectively protects against t-BHP-induced hepatotoxicity via inducing ERK/Nrf2-mediated antioxidative signaling pathway. Meanwhile, GA disturbs protein-protein interaction between Keap1 and Nrf2 which might also contribute to nuclear translocation of Nrf2.