RUFY3 is highly expressed in brain tissue and has a role in neuronal development. Transcriptional factor FOXK1 is involved in cell growth and metabolism. We knew that RUFY3 or FOXK1 has been correlated with the malignant of tumor cells. However, the role of these molecules in colorectal cancer (CRC) progression remains unknown. We investigated the protein expression levels by Western blot, immunofluorescence and immunohistochemistry analyses. The migration and invasive abilities of CRC cells were assessed using shRNA-mediated inhibition in vitro and in vivo. We showed that RUFY3 expression was up-regulated in CRC compared with its expression in a normal human colon cell line (FHC). RUFY3 suppression inhibited anchorage independent cell tumorigenesis. RUFY3 induced elevated expression of eight major oncogenes. Moreover, RUFY3 physically interacts with FOXK1 in CRC. A positive correlation was observed between the expression patterns of RUFY3 and FOXK1. Furthermore, RUFY3 and FOXK1 expression were correlated with tumor progression and represented significant predictors of overall survival in CRC patients. SiRNA-mediated repression of FOXK1 in RUFY3-overexpressing cells reversed the epithelial-mesenchymal transition (EMT) and metastatic phenotypes. In vivo, FOXK1 promoted RUFY3-mediated metastasis via orthotopic implantation. These findings suggest that the RUFY3-FOXK1 axis might promote the development and progression of human CRC.