Umami peptides can supplement and enhance the overall taste of food, making it more harmonious, soft, and full-bodied. Stropharia rugoso-annulata mushroom is famous for its umami and pleasant flavor, however, there were no report on its umami peptides. In this study, umami peptides from S. rugoso-annulata were isolated and characterized, as well as to analyze the action mechanism in silico molecular docking approach. By ultrafiltration, gel filtration chromatography, HPLC, and UHPLC-Q-Orbitrap-MS2, five peptides, EPLCNQ, SGCVNEL, PHEMQ, SEPSHF, and ESCAPQL were identified. According to the sensory evaluation and electronic tongue analysis, they had good umami activity and their umami threshold values were in the range of 0.167–0.390 mmol/L. EPLCNQ and SGCVNEL had higher umami intensity than others. Molecular docking of the identified peptides with the umami taste receptor T1R1/T1R3 indicated that the peptides can enter the binding pocket in the Venus flytrap domain of T1R3 cavity, wherein Glu45, Ser104, and His145 may play major roles in the binding interactions of peptides and receptor, forming more stable hydrogen bonds to producing strong umami taste. The study expands the source range of umami peptides and potential raw material of value-added umami-flavorings, and provides further insight into understanding the flavor mechanisms of mushrooms.