Colon cancer is a common cause of death in the world, and its main cause of therapy failure is chemoresistance. Apoptosis is de-regulated in colon cancer and is one key mechanism of cancer treatment. We recently reported that reduced expression of ARHGAP17, a Rho GTPase activating protein, correlated with a poor prognosis of colon cancer patients. Here we investigated the role of ARHGAP17 in apoptosis induced by 5-fluorouracil (5-FU) in human colon cancer cells and in mouse xenograft tumor model. We observed a decreased protein level of ARHGAP17 in 5-FU resistant colon cancer cells (HCT116/5-FU and HCT8/5-FU). While ARHGAP17 knockdown attenuated apoptosis upon 5-FU treatment in HCT116 and HCT8, and ARHGAP17 overexpression in HCT116/5-FU and HCT8/5-FU cells increased apoptosis induced by 5-FU. We also found that ARHGAP17 knockdown led to a high level of active Rac1 in HCT116 and HCT8, but ARHGAP17 overexpression reduced active Rac1 in HCT116/5-FU and HCT8/5-FU cells. However, Rac1 inhibitor abolished the effect of ARHGAP17 knockdown, and Rac1 overexpression diminished the effect of ARHGAP17 overexpression on apoptosis induced by 5-FU. Apoptosis was also confirmed by cleaved Caspase-3 and cleaved PARP. Further, we observed that overexpression of ARHGAP17 promoted 5-FU-induced apoptosis and attenuated tumor growth in vivo. Collectively, our data indicate that ARHGAP17 sensitizes chemotherapy-resistant colon cancer cells to apoptosis induced by 5-FU, which is in part through suppressing Rac1.