The secosteroid hormone vitamin D3 (VD3) exerts its biological actions through its cognate receptor, the vitamin D receptor (VDR). Vitamin D3 and VDR have a key function in bone formation and keratinocyte differentiation, exert antiproliferative actions in human cancer, and is widely used as a chemotherapeutic agent for cancer. In addition, VD3 promotes differentiation of human osteosarcoma cells by up-regulating genes involved in cell cycle arrest and osteoblastic differentiation. Although considerable work has been carried out in understanding the molecular mechanisms underlying the VD3-mediated differentiation of human osteosarcoma cells, the upstream regulation of VD3 signaling pathway is still unclear. In this study, we show that p73 acts as an upstream regulator of VD3-mediated osteoblastic differentiation. Transcription factor p73, a p53 homolog, has been shown to have a function in development and recently been termed as a tumor suppressor. Silencing p73 results in a significant reduction of VD3-mediated osteoblastic differentiation; although DNA damage induced p73 leads to an increase in VD3-mediated differentiation of osteosarcoma cells. Together, our data implicate a novel function for p73 in vitamin D-mediated differentiation of human osteosarcoma cells.