We determined the functional role of the Sertoli cell glucocorticoid receptor (GR) in vivo using a transgenic Cre-loxP approach to conditionally disrupt GR expression. Sertoli cell GR knock-out (SCGRKO) was shown by absent Sertoli cell-specific GR immunolocalization and reduced levels of glucocorticoid-responsive Stc1 and Tsc22d3 mRNA in SCGRKO relative to control testes. Adult SCGRKO testes exhibited distinct morphological changes, including reduced seminiferous tubular lumen formation, decreased total Sertoli cell numbers and parallel reductions in meiotic spermatocyte and postmeiotic spermatid numbers. Conversely, tubular diameter was increased and testis size was normal in SCGRKO males. Decreased serum FSH and testicular Fshr mRNA levels were consistent with reduced Sertoli cell number. Adult SCGRKO testes also displayed atypical germ cells and interstitial focal accumulations of hypertrophic lipid-laden, immature-like Leydig cells. Circulating LH, and testicular Lhr mRNA, testosterone, dihydrotestosterone and 3α/3β-diol levels were all reduced in mature SCGRKO mice, whereas serum testosterone and dihydrotestosterone levels remained normal. Moreover, Sertoli cell GR disruption caused differential changes to steroidogenic enzyme transcripts, with downregulated testicular Cyp11a1 contrasting with upregulated Hsd17b3 expression. Reduced SCGRKO testicular expression of Hsd11b2, encoding an enzyme for corticosterone inactivation, supports a dynamic coupling between Hsd11b and androgen production. Our novel SCGRKO model has revealed that Sertoli cell-mediated GR actions support normal testicular function. Sertoli cell GR is required to maintain normal testicular Sertoli/germ cell numbers and circulating gonadotrophin levels, as well as optimal Leydig cell maturation and steroidogenesis, providing new insight into gluocorticoid-mediated impact on male reproduction.