Abstract: Objective To investigate the expression of miR-133b in human gastric cancer cell lines, gastric mucosal epithelial cells in vitro, gastric cancer tissues and matched adjacent non-tumor tissues, and to explore the miRNA regulation role in the occurrence and development of gastric cancer. Methods We cultivated 7 gastric cancer cell lines and normal gastric mucosa epithelial cells, and collected 56 cases of gastric cancer and the corresponding para-carcinoma tissues, to extract the total Small RNA. After reverse transcription, real-time PCR was used to detect the expression of miR-133b in gastric cancer cells and tissues, to analyze its association with clinicopathological features and cell differentiation. Results The expression level of miR-133b was down-regulated in human gastric cancer cells and tissues, with statistic difference. The down-regulated miR-133b expression was associated with cell differentiation, lymph node metastasis and TNM stage in gastric cancer patients(P＜0.05 )．Conclusion The expression of miR-133b is significantly decreased in gastric cancer cells and tissues. miR-133b may be involved in the tumorigenesis of gastric cancer as an anti-oncogene.