Background: miRNAs are critical post-transcriptional regulators of gene expression and key mediators of tumourigenesis. miR-501-5p is newly identified to be involved in the tumor progression, but its biological role and mechanism remain largely unknown. This study is aimed to study the role of miR-501-5p in the progression of gastric cancer. Methods: Real-time PCR analysis was used to determine miR-501-5p expression in gastric cancer cell lines, clinical tissues and 112 clinicopathologically characterized gastric cancer specimens. The role of miR-501-5p in maintaining gastric cancer stem cell like phenotype was examined by tumor-sphere formation assay and expression of stem cell markers. Luciferase reporter assay, cellular fractionation and western blot analysis were used to determined that miR-501-5p activated the wnt/beta-catenin signaling by directly targeting DKK1, NKD1 and GSK3 beta. Results: Herein, our results revealed that miR-501-5p was markedly upregulated in gastric cancer cell lines and clinical tissues. High miR-501-5p levels predicted poor overall survival in gastric cancer patients. Gain-of-function and loss-of-function studies showed that ectopic expression of miR-501-5p enhanced the cancer stem cell-like phenotype in gastric cancer cells. Notably, wnt/beta-catenin signaling was hyperactivated in gastric cancer cells that overexpress miR-501-5p, and mediated miR-501-5p-induced cancer stem cell-like phenotype. Furthermore, miR-501-5p directly targeted and suppressed multiple repressors of the wnt/beta-catenin signaling cascade, including DKK1, NKD1 and GSK3 beta. These results demonstrate that miR-501-5p maintains constitutively activated wnt/beta-catenin signaling by directly targeting DKK1, NKD1 and GSK3 beta, which promotes gastric cancer stem cell like phenotype. Conclusions: Taken together, our findings reveal a new regulatory mechanism of miR-501-5p and suggest that miR-501-5p might be a potential target in gastric cancer therapy.