Hypsizygus marmoreus is an edible variety of mushroom with a distinct umami taste, which might be contributed by amino acids and umami peptides. Umami peptides have been widely studied, except for those from Hypsizygus marmoreus. Therefore, the main purpose of this study was to find new umami peptides in it. After the mushrooms were enzymatically hydrolyzed, they were separated and characterized by UF, GFC, RP-HPLC, UPLC-Q-TOF MS/MS and the umami peptide EGTAG was obtained. Sensory evaluation found that EGTAG had distinct umami taste with thresholds of 8.26 mmol/L and 10.04 mmol/L in water and chicken consommé, respectively. Molecular docking results showed that Glu120, Ser142, Asp162 and Gln361 of T1R1/T1R3 may play critical roles. Hydrogen and hydrophobic interactions were the main binding forces between T1R1/T1R3 and the umami peptide. We proposed umami peptide from Hypsizygus marmoreus may be used as savory enhancer in the salt-reducing foods in the future. HYPOTHESES: Edible fungi generally have umami taste and umami peptides from edible fungi had been reported in the literature. Hypsizygus marmoreus belongs to edible fungi and has umami characteristics, so it can be inferred that Hypsizygus marmoreus contains umami peptides.