Forkhead box K1 (FOXK1) is a member of the FOX transcription factor family, which plays an important role in oncogenesis. However, the exact function and mechanism of FOXK1 in human colorectal cancers (CRCs) remain unclear. In the present study, we first screened for potential FOXK1 target genes by ectopically expressing FOXK1 in SW480 cells and examined the subsequent changes in the expression levels of major oncogenes using RT-PCR. We also evaluated the effects of FOXK1 regulation on growth and apoptosis. In addition, we investigated the biological impact of FOXK1 knockdown on CRC cells in vitro and in vivo. We found that FOXK1 overexpression increased the expression of multiple oncogenes in vitro. FOXK1 promoted serum-dependent and anchorage-dependent and -independent cell growth. Knockdown of FOXK1 induced G0/G1 cell cycle arrest in CRC cells. Moreover, FOXK1 suppression induced apoptosis and increased cell susceptibility to 5-fluorouracil (5-FU)-induced apoptosis. Furthermore, a xenograft model was established to explore FOXK1 shRNA-mediated tumorigenesis in vivo. A strong antitumorigenic effect of FOXK1-shRNA was enhanced when combined with 5-FU treatment. These findings implicate FOXK1 as a cell cycle and growth modulator that inhibits apoptosis in colon cancer cells. FOXK1-shRNA may serve as a novel and potent therapeutic agent, alone or with 5-FU, against colon cancer.