Obesity is an independent risk factor for cardiovascular disease (CVD) although the precise molecular mechanisms that link obesity to CVD are not understood. Recent studies suggest that factors secreted by adipose tissue may play an adverse role in cardiovascular health. We have previously demonstrated that the adipocytokine, leptin, promotes vascular calcification in apolipoprotein E - deficient mice and that this increase in calcification is associated with an increase in the expression of several osteoblast-specific markers within the vessel wall. In an effort to understand the mechanism by which leptin exerts these effects, we cultured primary bovine aortic smooth muscle cells (BASMCs) in the presence of 0 to 2 μg/ml leptin for up to 12 days. Osteogenic differentiation of BASMCs was determined by an increase in the expression of osteoblast-specific markers, and the induction of both alkaline phosphatase activity and mineralization. Consistent with previous studies, we found that treatment of BASMCs with leptin induced osteoblast differentiation in a dose-dependent manner. To investigate the underlying mechanism, we examined changes in the expression levels of key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway. We found that exposure to leptin induced the Erk 1/2-dependent inactivation of GSK-3β, through Ser9 phosphorylation, and a subsequent increase in the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a > 2-fold increase in GSK-3β activity, a decrease in the expression of the osteoblast-specific marker, osteopontin, and a significant decrease in leptin-induced alkaline phosphatase activity. Together, our results provide a possible mechanism by which elevated leptin concentrations, associated with obesity, promote osteoblast differentiation, and vascular calcification in vivo.