Cyclin-dependent kinase inhibitor 3 (CDKN3) has been reported to promote tumorigenesis. Since it is unclear whether CDKN3 participates in the development of human gastric cancer, this study assessed the association between CDKN3 expression and cell biological function and demonstrated the clinical significance and prognosis of CDKN3 in human gastric cancer. In this study, we found that CDKN3 showed a high expression in 35 paired human gastric cancer tissues and was correlated with poor patient survival, AJCC clinical staging, and recurrence. Silencing of CDKN3 in human gastric cancer cells can significantly reduce proliferation, migration, invasion, and adhesion abilities. Also, silencing of CDKN3 in human gastric cancer cells can induce G0-G1 cell cycle arrest and apoptosis. Detection of cell cycle marker expression showed that CDKN3 knockdown promotes cell cycle arrest by decreasing the expression of CDK2, CDC25A, CCNB1, and CCNB2 in human gastric cancer cells. The results of this study will help elucidate the oncogene function of CDKN3 in human gastric cancer.