MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Dysregulated expression of miRNAs is associated with several diseases, including cancer. In this study, we report that the expression of microRNA-101 (miR-101) is down-regulated in gastric cancer tissues and cells, and ectopic expression of miR-101 significantly inhibits cellular proliferation, migration and invasion of gastric cancer cells by targeting EZH2, Cox-2, Mcl-1 and Fos. Our animal study also indicates that miR-101 could potentially suppress tumour growth in vivo. Collectively, these results suggest that miR-101 may function as a tumour suppressor in gastric cancer, as it has an inhibitory role not only in cellular proliferation, migration and invasion in vitro, but also in tumour growth in vivo.