The N-myc downstream regulated gene 1 (NDRG1)/Cap43 is closely associated with cell differentiation, and its expression is induced by hypoxia and increasing intracellular calcium levels. Whether the NDRG1/Cap43 expression in cancer cells is a predictive marker of good or poor prognosis in patients, depends upon tumor types and differentiation status. In this study, we examined whether the NDRG1/Cap43 expression was involved in the differentiation of osteosarcoma cells, using three osteosarcoma cell lines, MG63, U2OS and SaOS2. The NDRG1/Cap43 expression in MG63 and U2OS was significantly enhanced by vitamin D3, which also induced the production of osteocalcin, a differentiation marker of osteoblasts. The knockdown of NDRG1/Cap43 using small interfering RNA also suppressed the production of osteocalcin and enhanced cell proliferation, accompanied by the suppression of p21 expression. Furthermore, the acquired invasiveness of osteosarcoma cells during the invasion in Matrigel resulted in the decreased expression of NDRG1/Cap43. On the basis of these results, our proposed role for NDRG1/Cap43 would be in the capacity of differentiation and invasion in osteosarcoma cells.