The effects of topically administered snake (Deinagkistrodon acutus) oil and its main fatty acid components on skin photodamage were explored. Epidermal thickness, mice body weight, antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase), inflammatory cytokines (tumor necrosis factor alpha and interleukin-6), skin histology, collagen content, and metalloproteinase-1 indicators were analyzed. The results show that topical application of both snake oil and its main fatty acids recovered ultraviolet B (UVB) irradiation induced antioxidant enzymes depletion, prevented metalloproteinase-1. Snake oil and its main fatty acids suppressed dermal infiltration of inflammatory cells and reduced inflammatory cytokines levels. Notably, there was no significant difference in the antioxidant activity but a significant difference in the anti-inflammatory activity between fatty acids and snake oil under the same dose. Finally, snake oil and its main fatty acids inhibited UVB-induced histological damage such as epidermal thickening, collagen fiber and elastic fiber destruction. Our study demonstrated for the first time in KM mice that snake oil exhibited prominent photoprotection activity by protecting the activity of antioxidant enzymes and inhibiting inflammatory factors, as well as reducing the generation of MMP-1. What's more, the main fatty acids in snake oil play an important role in preventing photo-damage especially in protecting antioxidant enzyme activity.