Autologous transplantation of spermatogonial stem cells (SSCs) is a promising approach to restore fertility in young cancer patients who often become infertile due to gonadotoxic treatment. Because only a small number of SSCs can be collected from a biopsy, in vitro expansion of these cells is necessary before auto-transplantation. However, residual spermatogenesis in these patients poses.a significant barrier to efficient colonization of transplanted SSCs. Here, we report that inhibition of retinoic acid signaling by WIN18446, an ALDH1A2 inhibitor, allows colonization of cultured SSCs in nonablated mature testes. Cultured SSCs successfully colonized mouse testes lacking Cldn11-a key molecule for maintaining the blood-testis barrier (BTB)-but failed to colonize wild-type testes, suggesting that the BTB is a major hurdle for colonization. Although WIN18446 treatment did not downregulate CLDN11, it induced a deficiency in CLDN3, possibly through enhancing BTB permeability, and allowed for complete spermatogenesis even in allogeneic recipient testes without immunosuppression. Microinsemination resulted in the production of allogeneic offspring. These results indicate that inhibition of the BTB by WIN18,446 is applicable in cases where endogenous SSCs prevent donor SSC colonization and further demonstrate the unique ability of cultured SSCs to colonize allogeneic testes.