Gallbladder cancer (GBC) is a highly malignant bile duct cancer with poor prognosis characterized by its insensitivity to chemotherapy. Emerging evidence indicates that cytoprotective antioxidation is involved in drug resistance of various cancers; however, the underlying molecular mechanisms remain obscure. Here, we demonstrated that atypical protein kinase C iota (aPKC iota) mediated reactive oxygen species (ROS) inhibition in a kinase-independent manner, which played a crucial role in tumorigenesis and chemoresistance. Mechanistically, we found that aPKC iota facilitated nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation, nuclear translocation and activated its target genes by competing with Nrf2 for binding to Kelch-like ECH-associated protein 1 (Keap1) through a highly conserved DLL motif. In addition, the aPKC iota-Keap1 interaction was required for antioxidant effect, cell growth and gemcitabine resistance in GBC. Importantly, we further confirmed that aPKC iota was frequently upregulated and correlated with poor prognosis in patients with GBC. Collectively, our findings suggested that aPKC iota positively modulated the Keap1 -Nrf2 pathway to enhance GBC growth and gemcitabine resistance, implying that the aPKC iota-Keap1-Nrf2 axis may be a potential approach to overcome the drug resistance for the treatment of GBC.