Objective: To isolate and culture bone marrow mesenchymal stem cells (BMMSCs) and study the calcification in vitro. Methods: Bone marrow was collected from femur and tibia bones of 2-month-old male SD rats and was seeded in cell-culture flasks. The morphology and ultrastructure of BMMSCs were analyzed with phase-contrast microscope and transmission electron microscope (TEM). Three flasks of second-passage cells were collected and counted with a hemacytometer. The growth curve was obtained. Alkaline phosphatase histochemical staining was performed with Gomori method. Calcification in vitro was stained with modified von Kossa method. Results: BMMSCs had a spindle shape, with irregular processes on cell surfaces. A large number of rough-surfaced endoplasmic reticuli and mitochondria were observed through TEM. Cell doubling time was 72 hours. ALP-stain-positive cells accounted for 10% to 15%. Calcification in vitro was obvious. Conclusion: The BMMSCs culture system is established. Through induction, these cells differentiate to cells with osteogenesis capability and may be used to provide cells for bone engineering.