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Hyla annectans is a tree frog living in the southwestern plateau area of China where there is strong ultraviolet radiation and long duration of sunshine. So their naked skin may possess chemical defense components that protect it from acute photo-damage. However, no such peptide or components has been identified till to date. In the current work, two novel peptides (FW-1, FWPLI-NH2 and FW-2, FWPMI-NH2) were identified from the skin of the tree frog. Five copies of FW-1 and four copies of FW-2 are encoded by an identical gene and released from the same protein precursor, which possess 167 amino acid residues. FW-1 and -2 can exert significant anti-inflammatory functions by directly inhibiting Ultraviolet B irradiation (UVB)-induced secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). They may achieve this function by modulating the UV-induced stress signaling pathways such as Mitogen-activated protein kinases (MAPK) and Nuclear Factor Kappa B (NF-κB). Besides, FW-1 and -2 showed potential antioxidant effects on epidermis by attenuating the UVB-induced reactive oxygen species (ROS) production through an unknown mechanism. Considering small peptides’ easy production, storage, and potential photo-protective activity, FW-1/2 might be exciting leading compounds or templates for the development of novel pharmacological agents for the suppression of UVB-induced skin inflammation. Moreover, this study might expand our knowledge on skin defensive mechanism of tree frog upon UVB irradiation.