Overall, in this project, we isolated and identified a novel peptide from Phyllomedusa burmeisteri, the insulinotropic effect of this novel peptide Phylloseptin‐PBu has been substantially studied, we confirmed the energy‐mediated KATP and Ca2+ channels, and the GLP‐1 receptor initiated PKA activation involvements in this effect, which has never been published from amphibian sources, our study continuously provide novel candidate and fundamental understanding for the treatment of T2DM in future. Here, GLP1R is linked to type 2 diabetes mellitus.