Objective: With the rising prevalence of diabetes worldwide, increased attention is focused on natural drug candidates that can treat diabetes with high efficacy but without undesired side effects. Lentinus edodes is an edible and medicinal fungus, whose polysaccharides are one of the main components that have been reported to have hypoglycemic ability. However, the detailed underlying hypoglycemic mechanism of Lentinus edodes polysaccharides is still unknown. In this study, we extracted and prepared a novel polysaccharide from Lentinus edodes, which was named LNT-1. Methods: The aim of this study was to evaluate the hypoglycemic effect of LNT-1 on mice with type 2 diabetes that was induced by a high fat and high sugar diet and streptozotocin. To explore the possible mechanism, metabolomics analysis based on UPLC-Q-TOF/MS and molecular methods were performed. Results: Data showed that treatment with LNT-1 could ameliorate the damage in diabetic mice, including physiological and biochemical indexes, oxidative parameters and histopathological changes. Moreover, 36 potential biomarkers were screened using metabolomics analysis based on UPLC-Q-TOF/MS. Among them, the main metabolic pathways were glycerophospholipid metabolism, arachidonic acid metabolism and arginine biosynthesis, thereby suggesting that oxidative stress may be involved in the occurrence of diabetes. Both the mRNA and protein expression of the Nrf2/HO-1 signaling pathway was upregulated after treatment with LNT-1, indicating that the hypoglycemic effect of LNT-1 may be related to the activation of the Nrf2/HO-1 pathway. Conclusion: LNT-1 may be a potential natural drug candidate for the prevention and treatment of diabetes by regulating the oxidative stress response. Our study aimed to provide new insights into the application of Lentinus edodes and its polysaccharide as a drug candidate and as an active ingredient in functional foods.