ATP1B3 encodes the β3 subunit of Na /K -ATPase and is located in the q22-23 region of chromosome 3. Na /K -ATPase participates in normal cellular activities but also plays a crucial role in carcinogenesis. In the present study, we found that expression of the β3 subunit of Na /K -ATPase was increased in human gastric cancer tissues compared with that in normal matched tissues and that this increased expression predicted a poor outcome. ATP1B3 expression was elevated at both the mRNA and protein levels in gastric cancer cell lines relative to those in a normal gastric epithelial cell line. Interestingly, ATP1B3 knockdown significantly inhibited cell proliferation, colony-formation ability, migration, and invasion and increased apoptosis in human gastric carcinoma cell lines. Additionally, knockdown induced cell cycle arrest at the G2/M phase. Furthermore, we demonstrated that ATP1B3 silencing decreased the expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and phosphorylated AKT (p-AKT), indicating that ATP1B3 regulates gastric cancer cell progression via the PI3K/AKT signalling pathway. Hence, the β3 subunit of Na /K -ATPase plays an essential role in the tumourigenesis of gastric cancer and may be a potential prognostic and therapeutic target for the treatment of gastric cancer.