Triosephosphate isomerase (TPI) is highly expressed in many types of human tumors and is involved in migration and invasion of cancer cells. However, TPI clinicopathological significance and malignant function in gastric cancer (GC) have not been well defined. The present study aimed to examine TPI expression in GC tissue and its biological functions. Furthermore, we investigated its downstream genes by gene chip technology. Our results showed that TPI expression was higher in gastric cancer tissues than adjacent tissues, although no statistical differences were found between TPI expression and clinicopathological factors. TPI overexpression in human gastric carcinoma cell line BGC-823 enhanced cell proliferation, invasion and migration, but did not change cell cycle distribution, while TPI knockdown suppressed proliferation, invasion and migration, induced apoptosis and increased G2/M arrest of human gastric carcinoma cell line MGC-803. Since the cell division cycle associated 5 (CDCA5) was identified as the one with the most decreased expression after TPI knockdown, we investigated its role in MGC-803 cells. The results showed that CDCA5 knockdown also inhibited proliferation, migration, induced apoptosis and increased G2/M arrest similarly to TPI knockdown. CDCA5 overexpression promoted MGC-803 cell proliferation, clone formation and migration abilities. These results indicated that TPI expression level might affect GC cell behavior, suggesting that both TPI and CDCA5 might be considered as potential tumor markers related with GC development and might be potential new targets in GC treatment.