Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. CaMKII is a Ca2+-activated serine/threonine kinase and it regulates the activity of target proteins by phosphorylation, Dlx5 transcription factor plays crucial roles in osteoblast differentiation. The expression of Dlx5 is regulated by several osteogenic signaling pathways from early stages of osteoblastogenesis. In addition, Dlx5 can be phosphorylated and activated by p38, suggesting that the function of Dlx5 can be also modulated by post-translational modification. Although CaMKII and Dlx5 both play crucial roles during osteoblast differentiation, the interaction between CaMKII and Dlx5 has not been investigated. In the Current study, we examined the effects CamKII on the function of Dlx5. We found that CaMKII phosphorylates Dlx5, and that CaMKII increases the protein stability and the osteoblastogenic transactivation activity of Dlx5. Conversely, a CaMKII inhibitor KN-93 decreased the osteogenic and transactivation activities of Dlx5. These results indicate that CaMKII regulates osteoblast differentiation, at least in part, by increasing the protein stability and the transcriptional activity of Dlx5. (c) 2009 Elsevier Inc. All rights reserved.