p38 MAPK has been shown to regulate osteoblast differentiation. Inhibition of this kinase with inhibitors or dominant negative mutant impedes osteoblast differentiation. Yet the molecular mechanism behind this regulation is not well understood. Here we provide evidence that the effect of p38 MAPK on osteoblast differentiation can be mediated by osterix, a transcription factor necessary and sufficient for osteoblast differentiation. Inhibition of p38 MAPK had minimal effects on differentiation of p53^-/- osteoblasts, which had sustained osterix expression. Inhibition of p38 MAPK down-regulated the expression of osterix at both protein and mRNA levels, but not other transcription factors involved in osteoblast differentiation. More importantly, this inhibitory effect could be significantly relieved in osteoblasts overexpressing osterix. Further experiments support that osterix expression is mainly controlled by BMPs existing in the culture medium, secreted by osteoblasts or provided by serum, and p38 MAPK plays a positive role in BMPs-induced osterix expression. These findings identify a novel mechanism by which p38 MAPK regulates osteoblast differentiation.