MicroRNAs (miRNAs) play crucial roles in the development and progression of human cancers, including gastric cancer. The discovery of miRNAs may provide a new and powerful tool for studying the mechanism, diagnosis, and treatment of gastric cancer. Here we show that miR-181a levels were significantly downregulated in gastric cancer tissues compared with the adjacent normal regions in 80 paired samples. Moreover, the lower levels of miR-181a were associated with the pM or pTNM stage in clinical gastric cancer patients. In addition, the ectopic expression of miR-181a in the gastric cancer cell line HGC-27 inhibited cell proliferation, cell migration, and invasion by directly interacting with the mRNA encoding the oncogenic factor Prox1. Taken together, our results indicate that miR-181a might act as a tumor suppressor in gastric cancer, which may provide a novel diagnostic and therapeutic option for human gastric cancer in the near future.