Osteoporosis is closely related to BMMSCs differentiation. Teriparatide promotes BMMSCs proliferation and differentiation with unclear mechanism. Both miR-339 and DLX5 are closely related to the differentiation of BMMSCs. Our study intends to assess the mechanism by how teripeptide promotes the proliferation and differentiation of BMMSCs. Rat BMMSCs were cultured and transfected with miR-339 inhibitor/NC and then treated with tripeptide (0, 10, 20, 50 nmol/L) followed by analysis of cell proliferation by CCK8 assay, alkaline phosphatase (ALP) activity, expression of miR-339, DLX5, Runx2 and OCN by real-time PCR, and DLX5 protein level by Western blot. miR-339 inhibitor transfection significantly decreased miR-339 expression, increased DLX-5 protein level, cell number, ALP activity and expression of osteogenic genes. Compared with 0 nmol/L group, 10, 20, 50 nmol/L group presented significantly increased cell number. With increased teriparatide concentration, cell number, ALP activity and expression levels of Runx2 and OCN was increased gradually and miR-339 and DLX5 expression was reduced. The luciferase activity in miR339 inhibitor and pmirGLO-DLX5-3′ UTR-wt-transfected cells was higher than cells transfected with miR-339 NC and pmirGLO-DLX5-3′ UTR-wt. Teriparatide promotes BMMSCs osteogenic differentiation by down-regulating miR-339 which targets DLX5 expression.