Ca2+/calmodulin-dependent protein kinase IIα (α-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the a isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of α-CaMKII by using trifluoperazine, KN93, and α-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of α-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that α-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of α-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.