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Ionizing radiation (IR) is one of the key contributors that cause male infertility by disturbing spermatogenesis. Lycopene, a carotenoid with strong antioxidant properties, was shown to protect against oxidative damage induced by IR in several experimental models. The present study was designed to explore the possible protective effects of lycopene against IR‐induced testicular damage in C57BL/6 mice. Mice were administered lycopene (20 mg/kg) by oral gavage for seven consecutive days prior to a single dose of whole‐body X‐ray irradiation (4 Gy, 1 Gy/min). We observed that lycopene remarkably augmented sperm motility and reduced sperm abnormalities in mice following IR exposure. Histopathological analyses also revealed that lycopene ameliorated the structural damage of seminiferous tubules and enhanced the regeneration of seminiferous epithelium following IR stress. Moreover, lycopene attenuated IR‐induced oxidative stress, as evidenced by a decreasing lipid peroxidation level and an increase in the antioxidant enzyme superoxide dismutase activity. In addition, lycopene reduced the γH2AX expression and the number of TUNEL‐positive cells in the germinal epithelium, as well as restoring the imbalance of Bax/Bcl‐2 expression induced by IR exposure. Furthermore, lycopene prevented mitochondrial membrane potential depolarization and ATP reduction and preserved the activities of mitochondrial complexes I‐IV in the testes of mice after exposure to IR. Lycopene also improved mitochondrial biogenesis in testes of mice exposed to IR, presenting as restored expressions of PGC‐1α, Nrf1, and Tfam. Taken together, our results suggest that lycopene alleviates IR‐induced testicular damage, and the underlying mechanism involves at least in part the inhibition of the mitochondrial apoptotic pathway and the maintenance of mitochondrial respiration and biogenesis. The beneficial effect of lycopene highlights the therapeutic potential of this plant‐derived antioxidant against impaired spermatogenesis and male infertility induced by IR. [ABSTRACT FROM AUTHOR]