Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.