Objective To explore the protective effect and mechanism of hydrogen (H₂) on ionizing radiation injury of mouse spermatogonia. Methods Mouse spermatogonia GC-1 cells were divided into 4 groups:control group, H₂ group, irradiation group and irradiation plus H₂ group. The cells in the irradiation group and the irradiation plus H₂ group were given single ⁶⁰Co γ ray irradiation with a cumulative dose of 8 Gy (dose rate 0.897 Gy/min). The cells in the H₂ group and the irradiation plus H₂ group were cultured in a H₂ cell culture system (75% H₂, 20% O₂ and 5% CO₂) for 1 h before irradiation. Cell counting kit 8 (CCK-8) and flow cytometry were used to detect the effects of irradiation and H₂ treatment on viability and apoptosis of GC-1 cells 24 h after irradiation. The 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and mitochondrial membrane potential JC-1 fluorescence probes were used to detect the effects of irradiation and H₂ treatment on intracellular reactive oxygen species (ROS) and mitochondrial membrane potential of GC-1 cells 2 h after irradiation. Western blotting was used to detect the effects of irradiation and H₂ treatment on the expression of mitochondrial apoptosis pathway proteins (B-cell lymphoma-associated protein x[Bax], cytochrome c[Cyt-c] and cleaved caspase 3[an activation product of caspase 3]) in GC-1 cells 24 h after irradiation. Results CCK-8 results showed that H₂ significantly increased the viability of GC-1 cells after irradiation (P<0.01), and flow cytometry showed that H₂ significantly reduced the apoptosis rate (P<0.01). The results of specific fluorescent probe staining showed that H₂ reduced the increase of intracellular ROS and inhibited the decrease of mitochondrial membrane potential after irradiation (P<0.01 or P<0.05). Western blotting results showed that H₂ inhibited the expression of mitochondrial apoptotic proteins (Bax, Cyt-c and cleaved caspase 3) in GC-1 cells after irradiation (P<0.01 or P<0.05). Conclusion H₂ can protect mouse spermatogonia from ionizing radiation injury of ⁶⁰Co γ ray irradiation by reducing ROS production, protecting mitochondrial membrane potential, and inhibiting mitochondrial apoptotic pathway.