Semaphorin 5A, a member of semaphorin family, was originally identified as axonal guidance factor functioning during neuronal development. Previously, we showed that the expression of semaphorin 5A might contribute to the metastasis of gastric cancer. However, its functional roles and mechanism(s) in invasion and metastasis of gastric cancer remain unclear. By using human gastric caner cell lines Parental SGC7901, SGC7901-siScrambled and SGC7901-siSema 5A, we found that semaphorin 5A significantly promoted the invasive and metastatic abilities of gastric cancer cell in vitro. Semaphorin 5A increased the expression of MMP9 by activating phosphorylated ErK1/2 in gastric cancer cell. Furthermore, MEK inhibitor PD98059 and MMP9 antibody (Ab) significantly inhibited in vitro invasive and metastatic abilities induced by semaphorin 5A. Taken together, the present work revealed a novel function of semaphorin 5A that the existence of semaphorin 5A could promote invasion and metastasis of gastric cancer by regulating MMP9 expression, at least partially, via the MEK/ERKs signal transduction pathway. Semaphorin 5A and its regulated molecules could be the potential targets for cancer therapy.