Reactive oxygen species (ROS) are involved in the deleterious effects of UV light on skin. The antioxidant defense system is considered to be crucial for protecting skin from ROS. Recently, we showed that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, reduced oxidative stress in UVB-irradiated keratinocytes. This study set out to determine whether topically applied FDP could exert protective effects against UV-induced skin damage in hairless mice. An in vitro skin permeation study using Franz-type diffusion cells showed that the amount of [¹⁴C]-FDP that diffused through the skin increased in a time-dependent manner, and about 3.5% of the applied FDP penetrated the skin after 24 h. Topical application of FDP (1%) preserved the endogenous antioxidant capacity of skin such as catalase and glutathione, which were significantly reduced after UVB irradiation without FDP. FDP also reversed the loss of catalase protein and prevented the accumulation of carbonylated proteins induced by UVB irradiation. These results provide evidence that topically administered FDP could penetrate into the skin and attenuate UVB-induced oxidative skin damage in hairless mice.