Penfluridol, a commonly used antipsychotic agent in a clinical setting, exhibits potential anticancer properties against various human malignancies. Here, we investigated the effect of penfluridol on the biological behavior of colorectal cancer (CRC) cells. Cell viability and clonogenic potential were detected by the cell counting kit‐8 and colony formation assay. The cell apoptosis and cell cycle distribution were quantified through flow cytometry. Caspase‐3 activity, glucose consumption, lactate production, and intracellular ATP levels were evaluated using the corresponding commercial detection kits. The protein levels of related genes were detected through western blotting. Mitochondrial membrane potential was detected using JC‐1 staining. A CRC xenograft tumor model was used to validate the antitumor activity of penfluridol in vivo. Penfluridol reduced cell survival and promoted apoptotic cell death effectively through the mitochondria‐mediated intrinsic pathway in a dose‐dependent manner. Furthermore, the process of glycolysis in HCT‐116 and HT‐29 cells was inhibited upon penfluridol treatment, as evidenced by the decrease in glucose consumption, lactate production, and intracellular ATP levels. Further mechanistic studies revealed that penfluridol influenced cell apoptosis and glycolysis in CRC cells by downregulating hexokinase‐2 (HK‐2). The proapoptotic effect and glycolytic inhibition‐induced by penfluridol were effectively reversed by HK‐2 overexpression. Consistent with in vitro results, penfluridol could also suppress tumor growth and trigger apoptosis in vivo. Penfluridol triggers mitochondrial‐mediated apoptosis and induces glycolysis inhibition via modulating HK‐2 in CRC and provides a theoretical basis to support penfluridol as a repurposed drug for CRC patients.