Objective To study the inhibitory effect of miR-340 on proliferation and β-catenin pathway of colon cancer cell via targeting leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) gene. Methods Human colon cancer cell lines HT29, SW480, LoVo and normal intestinal epithelial cell HIEC were cultured. The expression level of miR-340 was detected in these cells. SW480 cells were divided into the miR-NC group (transfected with miR-NC mimic), miR-340 group (transfected with miR-340 mimic), pcDNA3.1 group (transfected with pcDNA3.1 plasmid), pcDNA3.1+miR-340 group (transfected with pcDNA3.1 plasmid and miR-340 mimic), and pcDNA3.1-lgr5+miR-340 group (transfected with pcDNA3.1-lgr5 plasmid and miR-340 mimic). Proliferation viability A490 was detected by MTS assay, the expression level of miR-340 was detected by PCR, the expression levels of Lgr5, β-catenin and cyclinD1 were detected by western blot, and miR-340 targeting Lgr5 was verified by a double luciferase reporter gene assay. Results The expression levels of miR-340 in HT29, SW480 and LoVo cells were lower than that in HIEC cells, and the expression level of miR-340 in SW480 cells was the lowest. The level of A490, the expression levels of Lgr5, β-catenin, cyclinD1 and fluorescence activity of Lgr5 wild-type double luciferase reporter gene in the miR-340 group were lower than those in the miR-NC group. The level of A490, the expression levels of Lgr5, β-catenin, and CyclinD1 in the pcDNA3.1+miR-340 group were lower than those in the pcDNA3.1 group. The level of A490, the expression levels of Lgr5, β-catenin, and CyclinD1 in the pcDNA3.1-Lgr5+miR-340 group were higher than those in the pcDNA3.1+miR-340 group. Conclusion miR-340 can inhibit the proliferation of colon cancer cells, in which targeting Lgr5 gene and inhibiting β-catenin pathway could serve as a possible mol. mechanism.