Polysaccharides derived from Pleurotus eryngii possess various bioactive properties, including antioxidant, antidiabetic, anti-inflammatory, and immunomodulatory effects. In this study, polysaccharides were extracted from P. eryngii fruiting bodies and exposed to gamma irradiation at doses of 50 and 100 kGy, with a dose rate of 5 kGy/h. The surface morphology of the polysaccharide irradiated at 100 kGy exhibited numerous pores and a smaller flake structure compared to those irradiated at 50 kGy and the non-irradiated sample. 1H and 13C NMR spectra of all samples indicated that both irradiated and non-irradiated polysaccharides exhibited α- and β-configurations, with signals corresponding to C1–C5 clearly observed. HPLC analysis of the polysaccharides revealed that glucose (75.23%), galactose (4.96%), glucuronic acid (1.38%), ribose (0.94%), rhamnose (2.35%), and mannose (3.87%) are the main components. All polysaccharides demonstrated antioxidant activity, which increased with concentration. Both non-irradiated and irradiated polysaccharides exhibited antidiabetic effects, significantly reducing blood glucose levels, and restoring insulin level with superiority of irradiated polysaccharides. Additionally, they significantly elevated body weight, slightly reduced MDA levels, and markedly enhanced catalase activity in treated rats compared to diabetic controls. The antidiabetic effects of the polysaccharides were further confirmed by histopathological examination of the pancreas and liver sections from polysaccharide-treated diabetic rats. This suggests that irradiation, by reducing the molecular weight of polysaccharides, enhances their bioavailability and efficacy in modulating glucose metabolism.