Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into various cell types, including osteocytes, chondrocytes, adipocytes, myocytes, and tenocytes. However, the difficulty or failure in expanding the mouse MSCs in vitro greatly hampered important research in animal models. The OP9, a stromal cell line from mouse bone marrow, has hematopoietic supportive capacity. Here, we report that the OP9 has the immunophenotype (CD45(-), CD11b(-), FLK-1(-), CD31(-), CD34(-), CD44(+), CD29(+), Sca-1(+), CD86(-), and MHCII(-)) identical to canonical mouse MSCs. The expression of CD140a(+), CD140b(+), alpha-SMA(+) and Calponin(+) suggested the perivascular origin of OP9. Functionally, the OP9 had strong clonogenic ability and could be induced into osteocytes, chondrocytes and adipocytes. The lymphocyte transformation test (LTT) and mixed leukocyte reaction (MLR) showed that the OP9 could suppress T lymphocyte proliferation stimulated by nonspecific mitogens (PHA) or allogeneic lymphocytes (BALB/c T cells). Finally, the migration of OP9 could be efficiently induced by bFGF, IGF-1, IL-3, PDGF-BB, TGF-beta1 and TGF-beta3. In conclusion, the OP9 were bona fide MSCs, and such homogenous cell line will be helpful to delineate biological features of MSCs at the stem cell level.