Emerging evidence suggests that altered expression of microRNAs (miRNAs) is involved in cancer progression. However, the role of miR-125a-5p in gastric carcinogenesis remains unknown. Quantitative real-time PCR analysis revealed that the expression of miR-125a-5p was significantly decreased in >80% of gastric cancer tissues compared with their adjacent non-tumor tissues, and was markedly reduced in ~95% of intestinal-type gastric cancer tissues. The downregulated miR-125a-5p was significantly associated with gastric cancer metastasis. Ectopic expression of miR-125a-5p substantially inhibited the proliferation, migration and invasion activities of gastric cancer cells. Furthermore, forced expression of miR-125a-5p repressed the activity of a luciferase reporter carrying the 3'-untranslated (3'-UTR) region of E2F3, which was eliminated by mutation of the predicted miR-125a-binding site, indicating that E2F3 may be a potential target gene of miR-125a-5p. These data suggest that by targeting E2F3, miR-125a-5p may be important as a potential tumor suppressor gene in gastric carcinogenesis.