Objective To study the ability of microRNA-129-5p (microRNA-129-5p, miR-129-5p) to regulate Wnt5a (wingless-type MMTV integration site family, member 5A) and to inhibit the proliferation and metastasis of gallbladder cancer cells and its potential mechanisms. Methods RT-PCR was conducted to detect the expression level of miR-129-5p in gallbladder cancer cells NOZ, SGC-996, GBC-SD and normal bile duct epithelial cell line HIBEpic. We selected gallbladder cancer cells with low miR-129-5p expression for miR-129-5p mimic transfection, and divided them into the control group (NC group) and miR-129-5p overexpression group (miR-129-5p mimic group). MTT assay was performed to detect the proliferation ability of each group of cells, and Transwell method was used to detect the metastasis ability. Targetscan 7.1 prediction software and dual luciferase reporter gene test were used to detect the targeted regulation of miR-129-5p on Wnt5a gene. RT-PCR was conducted to detect the expression level of Wnt5a mRNA in gallbladder cancer cells NOZ, SGC-996, GBC-SD and NC groups, as well as the miR-129-5p mimic group. Gallbladder cancer cells were divided into the NC group, miR-129-5p mimic group, miR-129-5p overexpression and Wnt5a knockdown plasmid co-transfection group (miR-129-5p mimic+sh-Wnt5a group) and miR-129-5p overexpression and Wnt5a overexpression plasmid co-transfection group (miR-129-5p mimic+oe-Wnt5a group). MTT assay was used to detect the proliferation ability of each group of cells, and the Transwell method was used to detect the transfer ability. Western-blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins β-catenin, cyclinD1, cMYC and MMP2 protein in each group of cells. Results Compared with the normal bile duct epithelial cell line HIBEpic, the expression of miR-129-5p in the gallbladder cancer cell line was reduced, and the expression in the gallbladder cancer cell GBC-SD was the lowest. Compared with the NC group, the proliferation and metastasis ability of gallbladder cancer cells in the miR-129-5p mimic group were reduced. Targetscan 7.1 prediction software, dual luciferase reporter gene and qRT-PCR test showed that miR-129-5p regulated Wnt5a. Compared with HIBEpic, the expression of Wnt5a in gallbladder cancer cell lines were increased. Compared with the NC group, the proliferation and metastasis ability of gallbladder cancer cells in the miR-129-5p mimic group, miR-129-5p mimic+sh-Wnt5a group, and miR-129-5p mimic+oe-Wnt5a group were reduced, and the miR-129-5p mimic+sh-Wnt5a group was the lowest, while the miR-129-5p mimic+oe-Wnt5a group was the highest. Compared with the NC group, expressions of β-catenin, cyclinD1, cMYC and MMP2 proteins of gallbladder cancer cells in the miR-129-5p mimic group, miR-129-5p mimic+sh-Wnt5a group and miR-129-5p mimic+oe-Wnt5a group were all decreased, among which the miR-129-5p mimic+sh-Wnt5a group was the lowest, while the miR-129-5p mimic+oe-Wnt5a group was the highest. Conclusion miR-129-5p targets the expression of Wnt5a to inhibit the proliferation and metastasis of gallbladder cancer cells.