Objective：This study aims to investigate the effect and mechanism of S100 calcium binding protein A16 （S100A16） on migration of gastric cancer cells. Methods：Gastric cancer cell lines SGC-7901 and MGC-803 with stable overexpression of S100A16 were constructed by lentivirus infection . Wound healing assay and Transwell assay were used to analyze the migration of gastric cancer cells. Liposome transfection method was used to transfect the S100A16 siRNA into SGC-7901，and expressons of E-Cadherin and Vimentin were detected by Western blot. The interaction between S100A16 and zonula occludens 2（ZO-2） was detected by co-immunoprecipitation. The location of S100A16 and ZO-2 was detected by immunofluorescence. Results：S100A16 expression was higher in gastric cancer cells than in normal gastric epithelial cells. Cell lines with S100A16 overexpression were stably constructed. Overexpression of S100A16 promoted the migration of gastric cancer cells. E-Cadherin protein increased，S100A16 and Vimentin proteins decreased in SGC-7901 cells by transfection of S100A16 siRNA. S100A16 interacted with ZO-2 in SGC-7901 cells. Conclusion：Overexpression of S100A16 promotes the migration of gastric cancer cells. Its mechanism may be through the interaction of ZO-2.