Previous studies have shown that microRNA-31 (miR-31) functions as a tumor-suppressor in various types of cancer. In the present study we found that miR-31 was significantly downregulated in gastric cancer (GC) as determined by microRNA (miRNA) array screening analysis. Although aberrant expression of miR-31 has been found in different types of cancer, its pathophysiological effect and role in tumorigenesis still remain to be elucidated. In the present study, we detected miR-31 expression in both metastatic GC cell lines and tissues that are potentially highly metastatic by real-time polymerase chain reaction (PCR). Transwell and scratch healing assays were conducted to examine whether the ectopic expression of miR-31 could modify the invasion and migration abilities of GC cells in vitro. We found that miR-31 inhibited GC metastasis in a nude mouse xenograft model of GC. Luciferase reporter assays demonstrated that miR-31 could directly bind to the 3' untranslated region of RhoA and downregulate the expression of RhoA. Significant downregulation of miR-31 in 78 GC tissues and four GC cell lines was examined by real-time reverse transcription-PCR. Moreover, the decreased expression of miR-31 was demonstrated to be associated with lymph node metastasis, poor pT and pN stage, and invasion ability into lymphatic vessels as determined by the Mann-Whitney U test. We also found that miR-31 could inhibit cell invasion and migration abilities in vitro using the Transwell and scratch healing assays in BGC-823, SGC-7901, MGC-803 as well as AGS cells. Experiments in a nude mouse model revealed that miR-31 suppressed tumorigenicity in vivo. The luciferase activity assay and western blotting indicated that RhoA was the potential target of miR-31 in GC cells. Collectively, our results provide important evidence that the downregulation of miR-31 inhibited the invasion and migration abilities of GC cells through direct targeting of the tumor metastasis‑associated gene, RhoA. These findings suggest that miR-31 may be a promising therapeutic candidate in the treatment of GC metastasis.