BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze–thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze–thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze–thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5–10 days old) than in mature (6–12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in‐vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.