Objective To investigate the effect of microRNA-141 (miR-141) gene interference targeting the protein tyrosine phosphatase gene (PTEN) on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs). Methods miR-141 mimics, an miR-141 inhibitor, miR-141 mimics-NC, and an miR-141 inhibitor-NC plasmids were constructed and transfected into mouse BMSCs to form an over expression group, silent group, over expression control group, and silent control group, respectively. Mouse BMSCs in routine culture were used as a blank control group. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the bone differentiation ability of each component. The expression of miR-141 and PTEN pathway-related genes and proteins was detected in each group to verify whether miR-141 can target PTEN. Results Compared with the blank control and over expression control groups, the over expression group had lower expression of ALP, AKT, and GSK3β mRNA and protein; Runx2, Osterix protein, p-AKT and p-GSK3β; and significantly decreased size, number and density of calcified nodules but higher expression of miR-141 mRNA and PTEN mRNA and protein. Compared with the blank control and silent control groups, the silent group had higher expression of ALP, AKT, and GSK3β mRNA and protein; Runx2, Osterix protein, p-AKT, and p-GSK3β; and significantly increased calcified nodules but lower expression of miR-141 mRNA and PTEN mRNA and protein. The luciferase activity experiment verified that miR-141 could target and regulate PTEN. Conclusions miR-141 overexpression activated the PTEN signaling pathway and inhibited the osteogenic differentiation of mouse BMSCs, while miR-141 gene silencing downregulated PTEN and upregulated AKT and GSK3β expression, increased p-AKT and p-GSK3β, promoted the expression of osteogenic marker protein, and promoted the osteogenic differentiation of mouse BMSCs.