Osteopontin (OPN), a glycosylated phosphoprotein that binds calcium, is present in bone extracellular matrix and has been reported to modulate both mineralization and bone resorption. Targeted disruption in mice of the vitamin D receptor (VDR) or Runx2 results in marked inhibition of OPN expression in osteoblasts. In this study, we addressed possible cross-talk between VDR and Runx2 in regulating OPN transcription. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) or Runx2 stimulated OPN transcription (mouse OPN promoter -777/+79) 2-3-fold. However, coexpression of Runx2 and VDR in COS-7 cells and treatment with 1,25(OH)(2)D(3) resulted in an 8-fold induction of OPN transcription, indicating for the first time functional cooperation between Runx2 and VDR in the regulation of OPN transcription. In ROS 17/2.8 and MC3T3-E1 cells that contain endogenous Runx2, AML-1/ETO, which acts as a repressor of Runx2, significantly inhibited 1,25(OH)(2)D(3) induction of OPN transcription, OPN mRNA, and protein expression. Both a Runx2 site (-136/-130) and the vitamin D response element (-757/-743) in the OPN promoter are needed for cooperative activation. Chromatin immunoprecipitation analyses showed that 1,25(OH)(2)D(3) can enhance VDR and Runx2 recruitment on the OPN promoter, further indicating cooperation between these two factors in the regulation of OPN. In osteoblastic cells, Hes-1, a downstream factor of the Notch signaling pathway, was found to enhance basal and 1,25(OH)(2)D(3)-induced OPN transcription. This enhancement was inhibited by AML-1/ETO, an inhibitor of Runx2. Immunoprecipitation assays indicated that Hes-1 and Runx2 interact and that 1,25(OH)(2)D(3) can enhance this interaction. Taken together, these findings define novel mechanisms involving the intersection of three pathways, Runx2, 1,25(OH)(2)D(3), and Notch signaling, that play a major role in the regulation of OPN in osteoblastic cells and therefore in the process of bone remodeling.