S100A14 is a calcium-binding protein involved in cell proliferation and differentiation as well as the metastasis of human tumors. In this study, we characterized the regulation of S100A14 expression between biological signatures and clinical pathological features in gastric cancer (GC). Our data demonstrated that S100A14 induced the differentiation of GC by upregulating the expression of E-cadherin and PGII. Moreover, S100A14 expression negatively correlated with cell migration and invasion in in vitro and in vivo experimental models. Interestingly, S100A14 blocked the store-operated Ca2+ influx by suppressing Orai1 and STIM1 expression, leading to FAK expression activation, focal adhesion assembly and MMP downregulation. Taken together, our results indicate that S100A14 may have a role in the induction of differentiation and inhibition of cell metastasis in GC.