Objective: To detect the expression of miR-362-3p in gallbladder cancer and its functions. Methods: The expressions of miR-362-3p in the surgical specimens from 44 gallbladder cancer patients were determined by qRT-PCR, and the relations of miR-362-3p expression with clinicopathological characteristics and prognosis of the patients were analyzed. In gallbladder cancer cells after transfection with miR-362-3p mimics, the changes in proliferation, migration and invasion were examined by MTT colorimetry, wound healing assay, and Transwell assay respectively. The target gene of miR-362-3p was analyzed by bioinformatics analysis and dual luciferase reporter gene assay, and then was verified through recovery tests. Results: The expression of miR-362-3p was significantly decreased in gallbladder cancer tissue compared with tumor adjacent tissue (P<0.05). The decreased miR-362-3p expression was significantly associated with tumor TNM stage, lymph node metastasis and distant metastasis (all P<0.05). The overall survival rate in patients with low miR-362-3p expression was significantly lower than that in patients with high miR-362-3p expression (P<0.05). The abilities of proliferation, migration and invasion were significantly reduced in gallbladder cancer cells after transfection with miR-362-3p mimics (all P<0.05). Nemo like kinase (NLK) was found potentially to be the target gene of miR-362-3p. After transfection with NLK overexpression vectors, the above effects exerted by miR-362-3p mimics in gallbladder cancer cells were significantly reversed (all P<0.05). Conclusion: The expression of miR-362-3p is down-regulated in gallbladder cancer, and the down-regulated miR-362-3p expression may reduce the inhibition on its target gene NLK, and thereby promote the proliferation, migration and invasion of gallbladder cancer cells.