Deregulation of glycolysis is a common phenomenon in human colorectal cancer (CRC). In the present study, we reported that Hexokinase 2 (HK2) is overexpressed in human CRC tissues and cell lines, knockout of HK2 inhibited cell proliferation, colony formation, and xenograft tumor growth. We demonstrated that the natural compound, xanthohumol, has a profound anti-tumor effect on CRC via down-regulation of HK2 and glycolysis. Xanthohumol suppressed CRC cell growth both in vitro and in vivo. Treatment with xanthohumol promoted the release of cytochrome C and activated the intrinsic apoptosis pathway. Moreover, our results revealed that xanthohumol down-regulated the EGFR-Akt signaling, exogenous overexpression of constitutively activated Akt1 significantly impaired xanthohumol-induced glycolysis suppression and apoptosis induction. Our results suggest that targeting HK2 appears to be a new approach for clinical CRC prevention or treatment.