Objective To examine the expression of cyclophilin A (CypA) in gastric cancer tissues and cell lines, and to explore the effect and mechanism of CypA on the proliferation of gastric cancer cells. Methods Real-time quantitative PCR and Western blotting analysis were used to detect the expression of CypA in gastric cancer tissues, the corresponding adjacent tissues and gastric cancer cell lines. Small interfering RNAs targeting CypA were synthesized and transfected into the gastric cancer cell line MKN45. The effect of CypA-siRNA on endogenous CypA expression was observed; meanwhile, nonspecific siRNA control group and negative control group were also designed. Cell proliferation was measured with MTT assay in each group; cell cycle was detected with flow cytometry. The expression of PCNA, P21, P16, and Cyclin D1 gene, involved in cell proliferation, were also detected by real-time quantitative PCR and Western blotting analysis. Results CypA expression was significantly up-regulated in gastric cancer tissues compared with para-cancer tissues; and CypA expression in gastric cancer cell lines was also significantly higher than that in the gastric epithelial cell line GES-1, with the highest expression found in the poorly-differentiated gastric cancer cell line MKN45(P<0.05). The expression of CypA was significantly inhibited by the CypA-siRNA in MKN45 (P<0.05).MTT assay showed that the inhibition of CypA by CypA-siRNA significantly suppressed MKN45 cell proliferation (P<0.05). The ratio of G₀/G₁ phase cells was significantly increased in MKN45 cells after CypA-siRNA transfection, while the ratio of G₂/M phase was significantly decreased(P<0.05). In addition, the expression of PCNA, Cyclin D1 was significantly decreased at mRNA and protein levels when CypA was inhibited, and the expression of P21 was significantly increased (P<0.05). Conclusion CypA is overexpressed in gastric cancer cells; CypA gene can effectively promote gastric cancer cell proliferation by regulating some proliferation related genes.