The aim of the present study was to determine the effects of UV irradiation on the conversion of 7-dehydrocholesterol (7-DHC), which has been coated onto a polystyrene surface, to cholecalciferol (D3), and the resulting effect on the formation of vitamin D (1,25-D3) by MC3T3-E1 cells. The changes in gene expression of the enzymes regulating its hydroxylation, Cyp27b1 and Cyp27a1, were monitored as well as the net effect of the UV-treated 7-DHC coating on cell viability and osteoblast differentiation. MC3T3-E1 cells were found to express the enzymes required for synthesizing active 1,25-D3, and we found a dose-dependent increase in the production of both 25-D3 and 1,25-D3 levels for UV-activated 7-DHC samples unlike UV-untreated ones. Cell viability revealed no cytotoxic effect for any of the treatments, but only for the highest dose of 7-DHC (20 nmol per well) that was UV-irradiated. Furthermore, osteoblast differentiation was increased in cells treated with some of the higher doses of 7-DHC when UV-irradiated, as shown by collagen-I, osterix and osteocalcin relative mRNA levels. The conversion of 7-DHC to preD3 exogenously by UV irradiation and later to 25-D3 by MC3T3-E1 cells was determined for the optimum 7-DHC dose (0.2 nmol per well), i.e. 8.6 ± 0.7% of UV-activated 7-DHC was converted to preD3 and 6.7 ± 2.8% of preD3 was finally converted to 25-D3 under the conditions studied. In conclusion, we demonstrate that an exogenous coating of 7-DHC, when UV-irradiated, can be used to endogenously produce active vitamin D. We hereby provide the scientific basis for UV-activated 7-DHC coating as a feasible approach for implant therapeutics focused on bone regeneration.