BACKGROUND: The isolated and purified mammary stem cells from Holstein dairy cows have been induced to differentiate into nerve cells, adipocytes and osteoblasts, further enriching their multi-directional differentiation potential, which is of great significance for the study on mammary stem cells. OBJECTIVE: To investigate the osteogenic potential of mammary stem cells from Holstein dairy cows. METHODS: Mammary stem cells from Holstein dairy cows were isolated and purified, which were provided by the Key Laboratory of Biological Manufacturing of Inner Mongolia Autonomous Region. The fourth generation of mammary stem cells were cultured for 21 days in an osteogenic induction medium, and identified by alizarin red staining and RT-PCR. RESULTS AND CONCLUSION: After osteogenic induction, the cell edge was indistinct, and there were some granular calcified nodules in the cells. The induced cells were positive for alizarin red staining. The positive expressions of Alpl and Bglap in the induced osteoblasts were detected, indicating that the mammary stem cells from Holstein dairy cows have the potential to differentiate into osteoblasts.