Xizang plateau frog (Nanorana parkeri) captured in Lhasa, Tibet, China, solely lives in the subtropical plateau, where there is strong ultraviolet radiation and long duration of sunshine. Considering its harsh living environment, the frog's innate defense against microbes and environmental stress was investigated. In current study, three antimicrobial peptides (AMPs) were purified and characterized from the skin secretion of N. parkeri. The coding cDNA sequences were also cloned from the skin cDNA library of N. parkeri. By structural characterization, two peptides were identified belonging to Japonicin-1 family, and named as Japonicin-1Npa (FLLFPLMCKIQGKC) and Japonicin-1Npb (FVLPLVMCKILRKC). The third peptide isolated named Parkerin with a unique sequence of GWANTLKNVAGGLCKITGAA did not show similarity to any known amphibian AMPs. Multi-functions of three AMPs were examined (antioxidant, MCD, hemolytic etc). Their solution structures determined by CD and antimicrobial mechanisms investigated by SEM are very well consistent with their functional characters. Current result suggests that these novel multi-functional AMPs could play an important role in defending N. parkeri against environmental oxidative stress and pathogenic microorganisms, which may partially reveal the ecological adaptation of these plateau-living amphibians.