A human skin equivalent was prepared by culturing human keratinocytes on the surface of nylon filtration meshes containing human skin fibroblasts and by growing the epidermal cells at the air–liquid interface. This human skin equivalent model was used to mimic the photoproduction of vitamin D₃ in human skin. It was found that the concentration of 7-dehydrocholesterol and its photoconversion to previtamin D₃ and its subsequent thermal isomerization to vitamin D₃ in the human skin equivalent was essentially identical to that of human skin. The 7-dehydrocholesterol content in the skin equivalent and human skin was 2187 ± 296 and 2352 ± 320 ng/cm², respectively. The percentage of the major photoproducts of 7-dehydrocholesterol in the skin equivalent following ultraviolet B radiation (0.5 J/cm²) was 35% pre-vitamin D₃, 29% lumisterol, and 6% tachysterol; 30% remained as 7-dehydrocholesterol. Similarly, in human skin they were 36%, 29%, 7%, and 28%, respectively. After incubation at 37° C for 30 min, 11% and 12% of the previtamin D₃ had thermally isomerized to vitamin D₃ in the skin equivalent and human skin. In conclusion, compared with cultured keratinocytes or fibroblasts, the human skin equivalent model provides a superior in vitro system that better mimics the physiology and biochemistry of the photosynthesis of vitamin D₃ in human skin.