Dry samples of fruit bodies of Cordyceps militaris and Agariucs blazei, mycelia of Antrodia camphorata, and hot water extracts of Ganoderma tsugae were ground and irradiated with γ-rays at doses of 0, 2.5, 5, and 10 kGy. After γ-irradiation, the content of γ-aminobutyric acid, adenosine, cordycepin, and triterpenoids in medicinal mushrooms did not significantly change. Gamma-irradiation only affects A. camphorata mycelia polysaccharide content, and an increase in irradiation dose (0-10 kGy) significantly reduces its content (111.85-101.88 mg/g). The content of high molecular weight (2.7×105 Da) polysaccharide in G. tsugae by γ-irradiation with increasing doses gradually increased. Gamma-irradiation could significantly affect the content of vitamin D2 and ergosterol. However, ergosterol content in non-irradiated C. militaris, A. blazei, A. camphorata, and G. tsugae was 2.34, 1.31, 2.33, and 1.15 mg/g, and of that in 10 kGy γ-irradiated mushrooms was 2.75, 1.88, 2.19, and 0.94 mg/g, respectively. Summarily, γ-irradiation does change some medicinal mushrooms by the content of physiologically active substances. When using γ-irradiation sterilization to maintain the physiological activity of mushroom products, one should consider its impact on the physiologically active substances.