The aim of this study was to identify anti-obesity peptide from Allomyrina dichotoma (A. dichotoma) and investigate the biochemical signaling pathway. For that, A. dichotoma larvae was hydrolyzed enzymatically, and further purified by using tangential flow filtration and consecutive chromatographic method. Finally, anti-obesity peptide was obtained, and their sequences identified as Gln-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu (EIA10). EIA10 prevented adipocyte differentiation in vitro and was further investigated the mechanism underlying the effects in vivo. Our results indicated that EIA10 reduced body weight gain, organ weight and adipose tissue volume. Glucose tolerance and insulin resistance in high fat diet-fed obese mice significantly improved after EIA10 administration for four weeks. In addition, EIA10 significantly decreased TC and LDL, increased HDL, improved lipid metabolism, and downregulated mRNA and protein expression of transcription factors implicated in lipid adipogenesis. Taken together our results suggest that EIA10 from possessed potential for the treatment and prevention of obesity.