Background: Penfluridol (PF) is an FDA-approved antipsychotic drug that has recently been shown to have anticancer activity. However, the anticancer effects and underlying mechanisms of PF are not well-established in gallbladder cancer (GBC). Methods: The anticancer efficacy of PF on GBC was investigated via a series of cell functions experiments, including cell viability, colony formation, apoptosis assays, and so on. The corresponding signaling changes after PF treatment were explored by western blotting. Then, nude mice were utilized to study and test the anticancer activity of PF in vivo. Besides, glucose consumption and lactic production assays were used to detect the glycolysis alteration. Results: In this study, we discovered that PF greatly inhibited the proliferation and invasion ability of GBC cells (GBCs). The glucose consumption and lactic generation ability of GBCs were dramatically elevated following PF treatment. Additionally, we discovered that inhibiting glycolysis could improve PF's anticancer efficacy. Further studies established that the activation of the AMPK/PFKFB3 signaling pathway medicated glycolysis after PF treatment. We proved mechanistically that inhibition of AMPK/PFKFB3 singling pathway mediated glycolysis was a potential synergetic strategy to improve the anticancer efficacy of PF on GBC. Conclusions: By inhibiting AMPK, the anticancer effects of PF on GBCs were amplified. As a result, our investigations shed new light on the possibility of repurposing PF as an anticancer drug for GBC, and AMPK inhibition in combination with PF may represent a novel therapeutic strategy for GBC. Graphical