CDC28 protein kinase regulatory subunit 2 (CKS2) is a cyclin-dependent kinase subunit (CKS) family member that participates in cell cycle regulation. Few studies have investigated its involvement in gastric cancer. In this study, we focused on the clinical and biological significance of CKS2 over-expression in gastric cancer. The expression of CKS2 mRNA was studied by real-time quantitative reverse transcription polymerase chain reaction, and the expression status in each tumor was examined to varify whether any correlation existed with clinical and pathological factors. In addition, an immuno-histochemical study was performed in selected samples. Moreover, we examined the impact of CKS2-siRNA in a gastric cancer cell line. A significantly higher expression of CKS2 mRNA was found in tumor tissues compared to paired normal tissues (p<0.01). Immunohistochemical analyses led to similar results. The overall five-year survival rate was significantly higher in the low CKS2 expression group (59.9%) than in the high CKS2 expression group (23.9%) (p<0.01). Univariate and multivariate analysis showed that CKS2 expression status was an independent prognostic factor (relative risk, 1.41; 95% confidence interval, 1.01-1.97; p<0.05). Moreover, the inhibition of cellular proliferation by CKS2-siRNA was observed in a gastric cancer cell line. CKS2 is one of the gastric cancer-related genes that correlates with biological aggressiveness and poor prognosis of gastric cancer. Thus, CKS2 is a possible candidate gene for diagnosis, as well as targeted molecular diagnosis and therapy in gastric cancer.