Semaphorins and their receptors are abnormally expressed in various cancers, but little is known about the expression and function of semaphorin 3E (SEMA3E) and its receptor, plexin D1 (PLXND1), in gastric cancer development or metastasis. We evaluated SEMA3E and PLXND1 expression by quantitative RT-PCR in gastric tissues from 62 patients who underwent gastrectomy and analyzed the correlation between their expression and clinicopathological variables. To assess the function of SEMA3E, we generated human gastric cancer cell lines with suppressed or increased SEMA3E expression. The expression level of SEMA3E, but not PLXND1, was correlated with lymph node involvement and metastatic progression in gastric cancer. A significant association was observed between a high level of SEMA3E expression and poor differentiation or poor survival in the intestinal type of gastric cancer. SEMA3E knockdown in gastric cancer cells attenuated cell proliferation and metastatic ability in vitro and in vivo. Moreover, SEMA3E caused cell proliferation and anchorage-independent cell growth in the intestinal type of gastric cancer. These results suggested that SEMA3E is likely to be involved in the development of gastric cancer and might also be a therapeutic target for its treatment.