Deregulation of E2F1 activity is characteristic of gastric tumorigenesis, which involves in complex molecular mechanisms. microRNA is one of the post-transcriptional regulators for gene expression. Here, we report a member of miR-331 family, miR-331-3p, which was decreased in some kinds of malignancies. However, the biological function of miR-331-3p on gastric cancer is largely unknown. In this study, we screened the expressing levels of miR-331-3p and E2F1 in gastric cancer cell lines. We transfected precursor or inhibitor of miR-331-3p into gastric cancer cells. As results, miR-331-3p is down-regulated in all gastric cancer cell lines by real-time PCR. Over-expression of miR-331-3p blocked G1/S transition on SGC-7901 and AGS cell lines. Introduction of miR-331-3p dramatically suppressed the ability of colony formation and cell growth in vitro by interfering E2F1 activity. Our data highlight an important role of miR-331-3p in cell cycle control by targeting 3'-UTR of cell cycle-related molecule E2F1. We concluded that miR-331-3p is a potential tumor suppressor in gastric cancer. Restoring miR-331-3p in gastric cancer cells revealed potential application in gastric cancer therapy. (C) 2010 Published by Elsevier Inc.