It has been reported that collagen and elastin inhibit skin wrinkle formation. Skin elasticity is closely associated with the formation of wrinkles induced by UV exposure. In this study, we investigated the protective effects Pleurotus ostreatus (P. ostreatus) on UVB irradiation induced wrinkle in hairless mice. We evaluated for their free radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), the anti-elastase activities, and for their anti-matrix metalloproteinase-1 (MMP-1) activity in human skin fibroblast cells. In the wrinkle measurement and image analysis of skin replicas, the results showed that P. ostreatus significantly inhibited wrinkle formation caused by chronic UVB irradiation. These results suggest that P. ostreatus has anti-wrinkle activity.