Aim: To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. Methods: Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free or-minimum es-sential media plus 10% fetal bovine serum (dextran-coated charcoal stripped)supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and l0 mmol/Lβ-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 μmol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. Results: The treatment of HBMSC with NTG (0.1-10 μmol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evi-denced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 μmol/L) could not be abolished by either NG-nitro-L-arginine methyl ester, an antagonist of endothe-lial NOS, or 1400W, a selective blocker of inducible NOS activity. Conclusion: NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.