In this study, we characterized the role of HA117, a novel gene we previously identified, in drug resistance in vitro and in vivo. Briefly, CT26 cells expressing HA117 were obtained by infection with a recombinant adenovirus, and tested for drug sensitivity by methyl thiazolyl tetrazolium assay. In addition, the effect of HA117 on drug sensitivity was assessed in CT26 colon tumors transplanted into nude mice. In vitro, expression of HA117 increased the resistance of CT26 cells to 5-fluorouracil 2.75 times (P < 0.05). Similarly, expression of HA117 in subcutaneously xenografted CT26 colon cancer significantly lowered its sensitivity to 5-fluorouracil (P < 0.05). Collectively, the results suggest that HA117 regulates the development of drug resistance in CT26 cells in vitro and in vivo.