Objective To explore the promoting effect and mechanism of miR-381-3p on osteogenic differentiation of human periodontal membrane stem cells (hPDLSCs) in inflammatory environment. Methods hPDLSCs were isolated and cultured in vitro, and identified by flow cytometry. hPDLSCs cells were transfected with miR-381-3p and the target gene of miR-381-3p was analyzed with bifluciferase detection reagent. hPDLSCs cells were divided into control group, miR-NC group, miR-NC+LPS group, miR-381-3p inhibitor group, miR-381-3p inhibitor +LPS group, miR-381-3p simulator group and miR-381-3p simulator +LPS group. After the intervention, the osteogenic differentiation ability of hPDLSCs and the ALP, mRNA expression of miR-381-3p, TLR4, NF-κB and Runx2 in hPDLSCs were detected. Results The isolated cells fit the characteristics of mesenchymal stem cells. miR-381-3p had a potential binding site for TLR4 and had a targeted regulatory effect. Compared with the control group, the mRNA expressions of miR-381-3p, TLR4 and NF-κB in hPDLSCs cells in LPS group were increased, while the levels of ALP, mineralized nodule formation and Runx2 mRNA were decreased(P＜0.05) Compared with miR-NC group and miR-NC+LPS group, mRNA expressions of miR-381-3p, TLR4 and NF-κB in hPDLSCs cells of miR-381-3p inhibitor group and miR-381-3p inhibitor +LPS group decreased, while the levels of ALP, mineralized nodule formation and Runx2 mRNA were increased, and the changes of miR-381-3p inhibitor group were more significant(P＜0.05) Compared with other groups, the mRNA expressions of miR-381-3p, TLR4 and NF-κB in hPDLSCs cells of miR-381-3p mimics group and miR-381-3p mimics +LPS group were increased, while the levels of ALP, mineralized nodule formation and Runx2 mRNA were decreased, and the changes of miR-381-3p mimics+LPS group were more significant(P＜0.05) Conclusion In inflammatory environment, the expression of miR-381-3p increases in hPDLSCs. Transfection of miR-381-3p inhibitor can inhibit the activation of TLR4/NF-κB signaling pathway and promote the osteogenic differentiation of hPDLSCs