To study the effect of dihydrotestosterone on the osteogenic differentiation of bone marrow mesenchymal stem cells after chemokine-like receptor 1 (CMKLR1) gene knockout, dihydrotestosterone and N,N,N-trimethyl-γ-oxo-2-naphthalenepropanaminium, monoiodide were injected in mice and induced bone marrow mesenchymal stem cells in vitro. The results showed that osteogenic differentiation rate decreased after CMKLR1 gene knockout and increased when dihydrotestosterone was injected in wild type mice bone marrow mesenchymal stem cells. However, there was no significant change in CMKLR1 knockout mice. The result indicated that the effect of dihydrotestosterone on bone cells was affected by the deletion of CMKLR1.