Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25-dihydroxyvitamin D3 (25OHD3) to the active steroid lα,25-dihydroxyvitamin D3 (1,25OH2D3) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D3, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D3 to 1,25OH2D3 and examined the potential of vitamin D3 to induce 1,25OH2D3 associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D3 to 1,25OH2D3 in a dose-dependent manner. It was also demonstrated that administration of vitamin D3 leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines 0PC1 andOMC3T3-E1. These findings provide a two-dimensional culture foundation for future three-dimensional engineered tissue studies using the OPC1 cell line.