Background: Gentamicin induces testicular damage in association with oxidative stress explained by different mechanisms, including gene expression suppression of some antioxidant enzymes. The aim of the study:To investigate the potential protective effects of α-lipoic acid against gentamicin-induced testicular oxidative stress in terms of altered gene expression. Materials and methods: A parallel experimental study was conducted including fifty adult male albino rats. The animals were grouped into five. The control group received intraperitoneal NaCl 0.9%, while the other groups received 36.5mg/kg/day intraperitoneal gentamicin. Group-1 received gentamicin only, group-2 received gentamicin + intraperitoneal α-lipoic acid 100mg/kg/day, group-3 received gentamicin + intraperitoneal α-lipoic acid 200mg/kg/day, and group-4 received gentamicin + oral vitamin E 100mg/kg/day. All treatments were given for 14 days. The animals were euthanized in two halves on the 15th and 60th days. Testes were immediately removed, frozen, and evaluated for oxidative stress biomarkers and gene expression of antioxidant enzymes. Results: Gentamicin increased malondialdehyde by mean difference ± standard error of 115.57 ± 2.18, decreased total antioxidant capacity by -9.23 ± 0.27, and decreased catalase and superoxide dismutase activities by -1.89 ± 0.45 and -11.77 ± 2.86, respectively, compared to the control. Additionally, gentamicin downregulated gene expression of catalase, glutathione peroxidase, and superoxide dismutase. However, adding vitamin E or α-lipoic acid cured the oxidative stress and partially to completely upregulated the gene expression. Conclusion: Reversal of testicular oxidative and gene suppression associated with gentamicin could be achieved with α-lipoic acid co-treatment, which might be applied in human studies