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Objective: To examine whether functional spermatozoa can be obtained when a whole male gonadal tissue (testes, epididymides, and fat) isolated from neonatal mice is grafted underneath adult mouse testes. Methods: Neonatal (1-day-old) male gonadal tissue, isolated from enhanced green fluorescent protein (EGFP)-transgenic (Tg) mice (C57BL/6-Tg(ACTB-EGFP)1Osb/J), was inserted deep in the testis of a non-Tg recipient mouse through a tunica albuginea incision. Two months after transplantation, the fluorescent grafted tissues were retrieved from recipient mice. Results: Histological analysis demonstrated that epididymal architecture was well developed and that spermatogenesis in the testis occurred in 30–60% of each seminiferous tubule of all the grafted tissues examined. Interestingly, motile spermatozoa could be successfully retrieved from the portion corresponding to the cauda epididymis in 1 of the 7 transplants obtained. These obtained spermatozoa had transgenes and could support embryonic development when intracytoplasmic sperm injection was performed using frozen-thawed spermatozoa. Conclusion: This present technique will be useful for study in various biological fields including the rescue of Tg lines with lethal postnatal phenotypes and cloned animals that die immediately after birth.