Osteoblast differentiation of human mesenchymal stem cells (hMSCs) is stimulated by 1α,25‐dihydroxycholecalciferol [1α,25(OH)2D3] and 25‐hydroxycholecalciferol [25(OH)D3]; the latter's effects require intracellular hydroxylation to 1α,25(OH)2D3. Thus, hMSCs are both a source of and target for 1α,25(OH)2D3. Megalin is a transmembrane receptor for serum d‐binding protein (DBP) in kidney cells and is required for uptake of the 25(OH)D3‐DBP complex. We tested the hypothesis that megalin is required for D actions in hMSCs with cells from surgically discarded marrow for RT‐PCR, for effects of 25(OH)D3 and 1α,25(OH)2D3, for 1α,25(OH)2D3 biosynthesis, for osteoblastogenesis, and for small interfering RNA for megalin (si‐Meg) and control (si‐Ctr). In hMSCs with high constitutive megalin expression, both 1α,25(OH)2D3 and 25(OH)D3 stimulated osteoblastogenesis (P < 0.05), but only 1α,25(OH)2D3 did so in hMSCs with lower megalin (lo‐Meg, P < 0.001) or in si‐Meg cells (P < 0.05). In addition, 1α,25(OH)2D3 biosynthesis was significantly lower in lo‐Meg (46%, P = 0.034) and in si‐Meg (23%, P < 0.001) than each control. Leptin significantly stimulated megalin expression 2.1‐fold in lo‐Meg cells (P < 0.01). These studies show that megalin is expressed in hMSCs and is required for the biosynthesis of 1α,25(OH)2D3 and for the 25(OH)D3/DBP complex to stimulate vitamin D receptor targets and osteoblastogenesis.—Gao, Y., Zhou, S., Luu, S., Glowacki, J. Megalin mediates 25‐hydroxyvitamin D3 actions in human mesenchymal stem cells. FASEB J. 33, 7684–7693 (2019). http://www.fasebj.org