Sertoli cell tight junction (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo, using the gonadotrophin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). FSH actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH), with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wildtype Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3 but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent but claudin-11 had redistributed basally toward the TJs where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein towards the basal region of Sertoli cells by 2 days, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by 10 days. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by 2 days of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.