Objective To detect expression of miR-106a-5p in gastric cancer cells and gastric cancer tissue, and to analyze relationship between it’s expression with clinicopathologic characteristics, and in addition, to analyze its target genes and enriched pathway with bioinformatics method. Methods The expressions of miR-106a-5p in the different differentiation of gastric cancer cells AGS (well differentiation), MKN-28 (middle differentiation), HGC-27 (undifferentiation), MGC-803 (low differentiation), BGC-823 (low differentiation), MKN-45 (middle differentiation) and SGC-7901 (middle differentiation), the normal gastric mucosal epithelial cells GES-1, and the gastric cancer tissue and the corresponding adjacent tissue were detected by the real-time fluorescent quantitative PCR. Furthermore, the target genes of miR-106a-5p were predicted by using more than three softwares affiliated to mirWALK web database and the signal pathways of target genes were enriched by DAVID 6.7 software. Results The expressions of miR-106a-5p in the different differentiation degree of the gastric cancer cells (AGS, SGC-7901, MKN-45, MGC-803, BGC-823, and HGC-27) were up-regulated except the MKN-28 cell line as compared with the normal gastric mucosa cell line GES-1 (P<0.010 orP<0.001), and the expression of miR-106a-5p in the gastric cancer tissue was also up-regulated as compared with the corresponding adjacent tissue, the expression of miR-106a-5p in the gastric cancer tissue was associated with the lymph node metastasis or the invasion depth. The results of the bioinformatics analysis showed that the target genes of miR-106a-5p were enriched in the multiple signaling pathways associated with the cancer. Conclusion miR-106a-5p is a molecular marker of high expression in gastric cancer and a potential cancer gene associated with lymph node metastasis and invasion depth.