Background: The aberrant expression of microRNA-140-5p (miR-140-5p) has been described in gastric cancer (GC). However, the role of miR-140-5p in GC remains unclear. In this study, the prognostic relevance of miR-140-5p in GC was investigated and YES1 was identified as a novel target of miR-140-5p in regulating tumor progression. Methods: miR-140-5p level was determined in 20 paired frozen specimens through quantitative real-time PCR, and analyzed in tissue microarrays through in situ hybridization. The target of miR-140-5p was verified through a dual luciferase reporter assay, and the effects of miR-140-5p on phenotypic changes in GC cells were investigated in vitro and in vivo. Results: Compared with that in adjacent normal tissues, miR-140-5p expression decreased in cancerous tissues. The downregulated miR-140-5p in 144 patients with GC was significantly correlated with the reduced overall survival of these patients. miR-140-5p could inhibit GC cell proliferation, migration and invasion by directly targeting 3'-untranlated region of YES1. miR-140-5p could also remarkably reduce the tumor size in GC xenograft mice. Conclusions: miR-140-5p serves as a potential prognostic factor in patients with GC, and miR-140-5p mediated YES1 inhibition is a novel mechanism behind the suppressive effects of miR-140-5p in GC.