We investigated the photoprotective effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) both in vivo and in vitro, revealing its relationship with glutathione, a well-known antioxidant. We also probed into the possible mechanism of photoprotection of 1,25(OH)2D3 through immunohistochemical study for metallothionein (MT). At the same time, endogenous antioxidant effect of 1,25(OH)2D3 was examined. Survival of cultured human keratinocytes was decreased when the cells were irradiated with ultraviolet light-B (UVB) at doses above 30 mJ/cm2. But in the presence of 1,25(OH)2D3 (12 nM), the decrease of survival of keratinocytes by UVB was diminished. The formation of sunburn cells by UVB irradiation in the skin of ICR mice was inhibited by topical application of 1,25(OH)2D3, regardless of prior glutathione depletion. Immunohistochemical staining revealed that 1,25(OH)2D3 induced the expression of MT, a potent radical scavenger, mainly in the basal layer of ICR mice skin. 1,25(OH)2D3 neither inhibited peroxidation of plasma lipids nor interacted with superoxide, nor removed hydrogen peroxide as an antioxidant. These findings suggest that 1,25(OH)2D3 has photoprotective effect not related with glutathione or its endogenous antioxidant property. Rather, it could be attributed to 1,25(OH)2D3-induced MT and its capacity to prevent radical-related damage in UVB irradiation.