Objective: Impairment of spermatogenesis has been identified as an inevitable side effect of cancer treatment. Although estrogen treatment stimulates spermatogenic recovery from the impaired spermatogenesis by suppressing the intra-testicular testosterone (ITT) level, side effects of estrogen are still major impediments to its clinical application in humans. Soybeans are rich in genistein, which is a phytoestrogen that binds to estrogen receptors and has an estrogenic effect. We investigated the effects of genistein administration on ITT levels, testis weight, and recovery of spermatogenesis in rats treated with a chemotherapeutic agent, busulfan. Methods: Busulfan was administered intraperitoneally to rats, and then a GnRH agonist was injected subcutaneously into the back, or genistein was administered orally. Results: The weight of the testes was significantly reduced by the treatment with busulfan. The testis weight was partially restored after busulfan treatment by additional treatment with either the GnRH agonist or genistein. Busulfan also induced atrophy of a high percentage of the seminiferous tubules, but this percentage was decreased by additional treatment with either the GnRH agonist or genistein. Treatment with genistein was effective at suppressing and maintaining ITT levels comparable to that in the GnRH agonist group. Conclusion: Genistein effectively suppressed ITT levels and stimulated the recovery of spermatogenesis in rats treated with a chemotherapeutic drug. This suggests that genistein may be a substitute for estrogens, for helping humans to recover fertility after cancer therapy without the risk of side effects.