p38 MAPK is a conserved subfamily of MAPKs involved in inflammatory response, stress response, cell growth and survival, as well as differentiation of a variety of cell types. In this report we demonstrated that p38 MAPK played an important role in osteoblast differentiation using primary calvarial osteoblast, bone marrow osteoprecursor culture, and a murine cell line, MC3T3-E1. We found that p38 MAPK was activated as calvarial osteoblast differentiates along with extracellular signal-regulated kinases (ERKs). When p38 MAPK is inhibited with a specific inhibitor, the expression of differentiation markers, such as alkaline phosphatase and mineral deposition, were significantly reduced. MC3T3-E1 cells expressing dominant negative p38 MAPK also displayed signs of delay in ALP and mineral deposition. Differentiation of the bone marrow osteoprecursors was also impeded by the p38 MAPK inhibitor, justified by the same markers. Yet the inhibitory effects observed in calvarial osteoblasts and bone marrow osteoprogenitor cells could be partially prevailed by bone morphogenetic protein-2. Inhibition of ERKs with a specific drug did not significantly affect osteoblast differentiation even though ERK1/2 were also activated during osteoblast differentiation. These results taken together indicate that p38 MAPK, but not ERKs, is necessary for osteoblast differentiation.