We have characterized the in vivo actions of human wildtype FSH receptor overexpressed in Sertoli cells of transgenic mice (TgFSHRwt) compared transgenic overexpression of the human activated mutant FSHR*D567G (TgFSHR*D567G). Testicular TgFSHRwt expression significantly elevated specific FSH binding (>2-fold, P < 0.01) relative to non-transgenic testes, similar to increased FSH binding in TgFSHR*D567G testes. Isolated TgFSHRwt Sertoli cells exhibited higher FSH-stimulated cAMP levels compared to non-Tg or TgFSHR*D567G cells, but did not display the elevated FSH-independent basal cAMP levels found in TgFSHR*D567G Sertoli cells. Furthermore, Sertoli cell overexpression of TgFSHR*D567G but not TgFSHRwt allowed promiscuous cAMP responses to hCG (300 IU/mL) and TSH (30 mIU/ml), demonstrating increased constitutive signaling and altered glycoprotein hormone specificity via the intracellular D567G substitution rather than FSH receptor overexpression. Despite elevating Sertoli cell FSH-sensitivity, overexpression of TgFSHRwt had no detectable effect upon normal testis function, and did not stimulate Sertoli and germ cell development in testes of gonadotrophin-deficient hypogonadal (hpg) mice, in contrast to the increased meiotic and postmeiotic germ cell development in TgFSHR*D567G hpg testes. Increased steroidogenic potential of TgFSHR*D567G hpg testes was demonstrated by elevated Cyp11a1 and Star expression, which was not detected in TgFSHRwt hpg testes. Androgen-regulated and Sertoli cell-specific Rhox5 gene expression was increased in TgFSHR*D567G but not TgFSHRwt hpg testes, providing evidence of elevated LH-independent androgen activity due to mutant FSHR*D567G. Hence, transgenic FSH receptor overexpression in Sertoli cells revealed that the D567G mutation confers autonomous signaling and steroidogenic activity in vivo, as well as promiscuous glycoprotein hormone receptor activation, independently of FSH receptor overexpression alone.