Objective To investigate the role of autophagy in ischemia/reperfusion injury (I/RI) caused by testicular torsion in rats. Methods Forty healthy male SD rats were randomly assigned (1:1:1) to sham group, model (I/RI) group, I/RI+rapamycin (RAPA) group, or I/RI+3-methyladenine (3-MA) group. Left testicular torsion (720°, 1 h) reduction rat models were established in the latter 3 groups. The rats in the I/RI+RAPA group and I/RI+3-MA group were intervened with autophagy agonist RAPA and autophagy inhibitor 3-MA before modeling, respectively. At 12 h after testicular reduction, the left testis and epididymis of rats in each group were collected for sperm quality analysis. The histopathological changes of testicular tissue were observed by hematoxylin-eosin (H-E) staining. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) in testicular tissue were measured by kits. The level of apoptosis in testicular tissue was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). The expression levels of autophagy-related proteins (Beclin1 and p62) and apoptosis-related proteins (Bcl2, Bax, and cleaved caspase 3) in testicular tissue were detected by Western blotting. Results Compared with the rats in the sham group, the rats in the I/RI group had poorer sperm quality, loosened testicular varicocele arrangement, fewer and shed germ cells, significantly lower SOD activity, T-AOC level, and p62 and Bcl2 expression levels (all P<0.05), significantly higher MDA content and expression levels of Beclin1, Bax, and cleaved caspase 3 (all P<0.05), and more apoptotic germ cells. Compared with the rats in the I/RI group, the rats in the I/RI+RAPA group had better sperm quality, normalized testicular varicocele arrangement, significantly higher SOD activity, T-AOC level, Beclin1 and Bcl2 expression levels (all P<0.05), significantly lower MDA content, p62, Bax and cleaved caspase 3 expression levels (all P<0.05), and fewer apoptotic germ cells; the rats in the I/RI+3-MA group had poorer sperm quality, loosened testicular varicocele arrangement, significantly lower SOD activity, T-AOC level, Beclin1 and Bcl2 expression levels (all P<0.05), significantly higher MDA content, p62, Bax and cleaved caspase 3 expression levels (all P<0.05), and more apoptotic germ cells. Conclusion Activation of autophagy can improve the sperm quality and testicular histopathological damage in I/RI rats, alleviate oxidative stress, and inhibit germ cell apoptosis.