Glucocorticoids inhibit testicular function, and the glucocorticoid receptor (GR) is expressed in Sertoli cells. To determine the possible role of glucocorticoids in Sertoli cells, we used a transgenic (Tg) Cre-loxP approach to conditionally disrupt the GR in Sertoli cells. TgAMH.Cre mice combined with floxed GR mice produced males lacking GR expression in Sertoli cells (SCGRKO), confirmed by immunohistochemical detection. Testicular expression of the known GR-regulated Stc1 gene was significantly reduced (50%, qPCR) in adult SCGRKO vs WT mice. Testis and epididymal weights were normal in SCGRKO mice. However, seminiferous tubules exhibited morphological changes, such as increased tubular diameter (20%), and less tubules (40%) containing a lumen. In addition, total Sertoli cell numbers per testis (by stereology) were significantly reduced (25%) in adult SCGRKO mice. Total testicular meiotic pachytene spermatocyte and round spermatid numbers were also reduced, but elongated spermatid numbers were normal, and males were fertile. Serum FSH and LH levels were reduced by 20 and 53 %, respectively, in adult SCGRKO versus WT males. Despite reduced gonadotrophin levels, serum testosterone and dihydrotestosterone levels (LC-MS/MS), and androgen-dependent seminal vesicles weights were normal. In contrast, intratesticular testosterone, dihydrotestosterone and 3α/3β-diols were all significantly reduced in SCGRKO mice, despite normal Leydig cell numbers. Expression of Leydig cell steroidogenic transcripts showed differential changes in response to Sertoli GR loss, with reduced Lhr and Cyp11a1 mRNA levels contrasting with normal Hsd3b6 and elevated Hsd17b3 expression in SCGRKO versus WT testes. Transcripts for enzymes that inactive corticosterone were normal (Hsd11b1) or reduced (Hsd11b2) in SCGRKO testes, the later though to inhibit adverse effects of excessive corticosterone on testosterone production. These results show for the first time a selective role for Sertoli cell GR that impacts gonadotrophin levels and Sertoli cell numbers, as well as Leydig cell steroidogenesis.