Although APEX1 is associated with the tumorigenesis and progression of some human cancer types, the function of APEX1 in gallbladder cancer (GBC) is unclear. In this study, we found that APEX1 expression is up-regulated in GBC tissues, and APEX1 positive expression is related to aggressive clinicopathological features and poor prognosis of GBC. APEX1 was an independent risk factor of GBC prognosis, and presented some pathological diagnostic significance in GBC. Furthermore, APEX1 was overexpressed in CD133+GBC-SD cells in comparison with GBC-SD cells. APEX1 knockdown increased the sensitivity of CD133+GBC-SD cells to 5-Fluorouracil via facilitating cell necrosis and apoptosis. APEX1 knockdown in CD133+GBC-SD cells dramatically inhibited cell proliferation, migration, and invasion, and promoted cell apoptosisin vitro. APEX1 knockdown in CD133+GBC-SD cells accelerated tumor growth in the xenograft models. Mechanistically, APEX1 affected these malignant properties via upregulating Jagged1 in CD133+GBC-SD cells. Thus, APEX1 is a promising prognostic biomarker, and a potential therapeutic target for GBC.