Curcumin, the major pigment of the dietary spice turmeric, has the potential for chemoprevention by promotion of apoptosis. Here, we investigated the molecular mechanisms of curcumin in glycolytic inhibition and apoptotic induction in human colorectal cancer HCT116 and HT29 cells. On the one hand, curcumin downregulated the expression and activity of hexokinase II (HKII) in HCT116 and HT29 cells in a concentration-dependent manner, but had little effect on the other key glycolytic enzymes (PFK, PGM, and LDH). On the other, curcumin induced dissociation of HKII from the mitochondria, resulting in mitochondrial-mediated apoptosis. Furthermore, the phosphorylation of mitochondrial HKII through AKT was responsible for the curcumin-induced dissociation of HKII, which was different from the mechanism of HKII inhibitor 3-BrPA. These results have important implications for the metabolism reprogramming effect and the susceptibility to curcumin-induced mitochondrial cytotoxicity through the regulation of HKII, and provide a molecular basis for the development of naturally compounds as novel anticancer agents for colorectal carcinoma.