There are many human extra-renal tissues and cells that biosynthesize 1?,25-dihydroxyvitamin D (1?,25(OH)2D) by the action of CYP27B1/1?-hydroxylase. Human marrow stromal cells (hMSCs), also known as mesenchymal stem cells, were isolated from marrow discarded from well-characterized, consented subjects during common orthopedic procedures. Human MSCs can give rise to osteoblasts, chondrocytes, adipocytes, and other lineages. Their in vitro differentiation to osteoblasts is stimulated by 1?,25(OH)2D, and recent evidence indicates that they have the capacity to metabolize vitamin D in a regulated manner. Human MSCs express the vitamin D receptor, 25-hydroxylases, 1?-hydroxylase, and 24-hydroxylase; stimulation of in vitro osteoblastogenesis by 25(OH)D depends on the activity of CYP27B1/1?-hydroxylase. The finding that hMSCs are a both a producer and target of 1?,25(OH)2D suggests a potential autocrine/paracrine role of vitamin D metabolism in osteoblast differentiation. Expression and enzyme activity of CYP27B1/1?-hydroxylase are upregulated by substrate 25(OH)D and Parathyroid Hormone (PTH) and are downregulated by 1?,25(OH)2D. With subject age, there are decreases in basal osteoblast potential and in stimulation of osteoblastogenesis by 1?,25(OH)2D, 25(OH)D, and PTH. In vitro treatment with a combination of 25(OH)D and PTH rejuvenated osteoblastogenesis with hMSCs from elders; this was attributable to increases in CYP27B1/1?-hydroxylase and in receptor for each hormone by the reciprocal factor. Other clinical variables beside age, i.e. low serum 25(OH)D or low estimated glomerular filtration rate, are correlated with reduced osteoblastogenesis. These studies suggest that osteoblastogenesis may not be optimal unless there is sufficient serum 25(OH)D substrate for hMSCs to synthesize and respond to local 1?,25(OH)2D.