The role of tumor suppressors and cell cycle factors in gastric carcinogenesis are well understood; however, the post-transcriptional changes that affect gene expression in gastric cancer are poorly characterized. MiR-135a has been shown to play a role in Hodgkin lymphoma. The aim of this study was to investigate the expression and role of miR-135a in gastric cancer. Quantitative real-time PCR demonstrated that miR-135a expression is downregulated in the majority of human primary gastric cancer tissues (8/11; 73%), compared with pair-matched adjacent non-tumor tissues. Furthermore, compared with the nonmalignant gastric cell line, GES-1, miR-135a expression was substantially downregulated in gastric cancer cell lines of various degrees of differentiation. Target analysis indicated miR-135a directly regulates Janus kinase 2 (JAK2), a cytoplasmic tyrosine kinase involved in cytokine receptor signaling pathways. Overexpression of miR-135a significantly downregulated the expression of JAK2 protein and also reduced gastric cancer cell proliferation and colony formation in vitro. MiR-135a-mediated JAK2 downregulation also reduced p-STAT3 activation and cyclin D1 and Bcl-xL protein expression. This study suggests that miR-135a may function as a tumor suppressor via targeting JAK to repress p-STAT3 activation, reduce cyclin D1 and Bcl-xL expression and inhibit gastric cancer cell proliferation. These results imply that novel treatment approaches targeting miR-135a may potentially benefit patients with gastric cancer.