Colorectal cancer is one of the most severe subtypes of cancer, and has the highest propensity to manifestas metastatic disease. Because of the lack of knowledge of events that correlate with tumor cell migration andinvasion, few therapeutic options are available. The current study aimed to explore the mechanism of colorectalcancer in hope of identifying the ideal target for future treatment. We first discovered the pro-tumor effect ofa controversial cell cycle regulator, cylin-dependent kinase inhibitor 3 (CDKN3), which is highly expressed incolorectal cancer, and the possible related signaling pathways, by bioinformatics tools. We found that CDKN3had remarkable effects in suppressing colorectal cancer cell proliferation and migration, inducing cell cyclearrest and apoptosis in a colorectal cancer cell line, SW480 cells. Our study, for the first time, provided consistentevidence showing overexpression of cell cycle regulator CDKN3, in colorectal cancer. The in vitro studies inSW480 cells revealed a unique role of CDKN3 in regulating cellular behavior of colorectal cancer cells, andimplied the possibility of targeting CDKN3 as a novel treatment for colorectal cancer.