Ca2+/calmodulin-dependent protein kinase II alpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue but it is becoming increasingly evident that CaMKII is also expressed in various extra-neural 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 demonstrate that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by calmodulin antagonist, TFP or the CaMKII antagonist, KN93 reduces alkaline phosphatase (ALP) activity and mineralization, as well as causing 85% and 56% decreases in ALP 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 alpha-CaMKII by using TFP, KN93 and alpha-CaMKII siRNA decrease the phosphorylation of ERK and of CREB leading to a significant decrease in the transactivation of SRE and CRE. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation and AP-1 DNA binding activity. Our findings demonstrate that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of SRE and CRE. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.