Ginkgo biloba leaf extract (GIN) is a popular Chinese herbal medicine. It has a nephroprotective effect against the nephrotoxicity induced by the chemotherapeutic agent methotrexate (MTX). This work was designed to explore the testicular protective role of GIN on MTX-induced testicular injury in a rat model. The experimental protocol lasted for 10 days for the 4 studied groups. First group: received saline (normal control, NC group). The second group was administered GIN (100 mg/kg/day) orally for 10 days (GIN C). Third group: injected with MTX (20 mg/kg ip) only on the fifth day (MTX group). Fourth group: administered GIN for 10 days with MTX injection on the fifth day (GIN+MTX group). MTX induced testicular injury as evident by a marked rise in the malondialdehyde (MDA) content, interleukin-6 (IL-6) and IL-1β protein levels, nuclear factor kappa-B (NF-κB) protein expression, bcl-2 associated × protein (Bax) mRNA expression, p53 mRNA and protein expressions, and miRNA29-a expression along with a marked decline in the serum level of testosterone and superoxide dismutase (SOD) content in testicular tissue in relation to the NC group. Moreover, histopathological testicular damage with a notable decrease in the Johnsen score together with a significant elevation in the testicular injury score was observed in the MTX group in comparison to the NC group. The administration of GIN ameliorated the biochemical changes as well as the testicular histopathological findings and scores. GIN could protect against MTX-induced gonadotoxicity by its antioxidant, anti-inflammatory, antiapoptotic activities plus the regulation of the miRNA-29a testicular expression.