The transmembrane protease, serine 3 (TMPRSS3), a member of the type II transmembrane serine protease family, plays an important role in mediating tissue development, homeostasis and various biological processes. Recently, TMPRSS3 has been reported to be involved in cancer progression. However, the role of TMPRSS3 in gastric cancer (GC) remains largely unknown. In this study, we found that TMPRSS3 was highly expressed in GC tissues and cell lines. Knockdown of TMPRSS3 inhibited GC cell proliferation, invasion and epithelial-mesenchymal transition (EMT) in vitro as well as suppressed GC cell growth and dissemination in vivo. These inhibitory effects were mediated by regulation of the ERK1/2 signaling pathway. Moreover, TMPRSS3-mediated ERK1/2 activation was dependent on the PI3K/Akt pathway. In conclusion, TMPRSS3 contributed to GC progression via activation of the PI3K/Akt/ERK signaling pathway and might act as a therapeutic target for GC treatment.