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This study evaluated the long-term protective effect of gallic acid (GAL) against testicular lesions induced by busulfan (BUSLF) in Wistar rats. Thirty (30) male rats weighing 60-70 g were randomized into three groups of ten in each group: control (2 ml kg -1 body weight (b.w) olive oil), BUSLF (10 mg kg -1 b.w), and BUSLF + GAL (10 mg kg -1 b.w BUSLF + 20 mg kg -1 b.w GAL). BUSLF was injected (intraperitoneally) concurrently with GAL (oral gavage) on day 1 but GAL administration continues for 12 weeks in the BUSLF + GAL animals. At the end of the study, all animals did not show relevant changes in body weights, but absolute testis weight and gonado-somatic index were decreased in the BUSLF-treated animals compared to the control values (p < 0.05). These biometric data remained unchanged in the BUSLF + GAL group relative to the control but were higher than the BUSLF values (p < 0.05). GAL co-treatment counteracted BUSLF-induced decrease in glutathione peroxidase activity and an increase in hydrogen peroxide, malondialdehyde, and carbonyl protein concentrations in the testis. Changes in testicular sorbitol and lactate dehydrogenases and myeloperoxidase activities in BUSLF-treated animals were ameliorated in the BUSLF + GAL-treated animals. GAL co-treatment also prevented BUSLF-induced decrease in testosterone and sialic acid concentrations and sperm quality. The spermatogenesis score index and histological changes induced by BUSLF were also abated in the BUSLF + GAL group. GAL has been established as an effective treatment regimen for the gonadal side effects of BUSLF in a rat model., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)