Objective To investigate the role of autophagy in the apoptosis of GC-2 spermatocytes in mouse induced by ionizing radiation. Methods The mouse spermatocytes GC-2 cells were divided into control group and 2, 4 and 8 Gy ⁶⁰Co irradiation treatment groups. The cell apoptosis was detected by in situ terminal transferase labeling (TUNEL) method and flow cytometry, the changes of autophagosome in GC-2 cells was observed by fluorescence microscope, and the expressions of autophagy-related proteins LC3 (LC3-Ⅰ and LC3-Ⅱ) and Beclin1 in GC-2 cells were determined by Western blotting analysis. After treatment with autophagy inhibitor 3-methyladenine (3-MA) 2 h before ionizing radiation treatment, the effect of autophagy inhibitor combined with ionizing radiation on cell viability and the changes of autophagy-related protein expressions in GC-2 cells were observed. Results Compared with the control group, the apoptosis rate and the expression of LC3-Ⅱ and Beclin1 protein of GC-2 cells in the irradiation treatment group were significantly increased (P<0.05). Fluorescence microscopy showed that the cell autophagosome was increased. The expression of Beclin1 and LC3-Ⅱ protein in GC-2 cells treated with 5 mmol/L 3-MA was significantly lower than that in the 3-MA-untreated group (P<0.05), and the apoptotic rate was significantly increased (P<0.05). Conclusion Ionizing radiation can induce autophagy of spermatocytes, and the inhibition of autophagy can enhance the killing effect of ionizing radiation on spermatocytes.