Drug combination is considered to be an effective approach to improve the efficacy of cancer therapy and chemoprevention. Selenite, a representative of inorganic form of selenium, and butyrate, a major short-chain fatty acid, are two well-documented colon cancer dietary chemopreventive agents with distinct molecular mechanisms. We hypothesized that combination of selenite and butyrate might produce improved outcome against colon cancer. This hypothesis was tested using both HCT116 human colon cancer cells and its xenograft mouse model in the present study. The in vitro study showed a synergistically inhibitory effect on HCT116 colon cancer cells but not on NCM460 normal human colon mucosal epithelial cells. Consistent with the in vitro study, results of the xenograft mouse model further demonstrated that combination of selenite and butyrate led to improved efficacy in comparison with each agent alone. Mechanistically, the induction of alanine-serine-cysteine transporter 2 (ASCT2) by selenite repressed its inhibitory effect on colon cancer cells, which was reversed by its co-treatment with butyrate. The findings of the present study denote the likely potential for developing selenite/butyrate combination remedy to combat against colon cancer.