The effects of high-energy electron beam irradiation on the physicochemical properties and antifatigue activity of cultivated Ganoderma lingzhi polysaccharides (GLPs) were investigated. GLPs were extracted using the water extraction and alcohol precipitation method and subsequently purified through ion exchange and gel filtration chromatography. Different doses of high-energy electron beam irradiation were applied to GLPs, and their physicochemical properties were characterized using scanning electron microscopy and infrared spectroscopy. A rat model of exercise fatigue was established through weight-loaded swimming, and GLPs were administered via gavage to verify their anti-fatigue effects. The results showed that the weight-average molecular weight of GLPs was 1.87×106, with an average particle size of 779.42 nm, and the anomeric carbon was in the β-configuration. As the irradiation dose increased, the molecular weight and particle size of GLPs initially increased and subsequently decreased, indicating a transition from cross-linking to depolymerization. GLPs treated with 20 kGy irradiation was found to significantly increase the liver and muscle glycogen contents (by 31.37% and 52.94%, respectively) while decrease the blood urea nitrogen and serum lactic acid contents (by 41.67% and 43.75%, respectively) in fatigued rats. Additionally, the irradiated GLPs enhanced the endogenous antioxidant enzyme activity in rat mitochondria and reduced malondialdehyde accumulation. Compared with the positive control (Rhodiola rosea glycoside), GLPs treated with 20 kGy irradiation demonstrated superior anti-fatigue effects. Therefore, an appropriate dose of highenergy electron beam irradiation was deemed an ideal method for modifying GLPs.