The influence of thyroid hormone (TH) on male reproduction is not well understood. Male mice lacking the type 3 deiodinase (DIO3), which inactivates TH, exhibit deficits in testis growth, reproductive hormone levels, spermatogenesis and fertility (Martinez et al, Endocrionology 157:1276, 2016). We aimed to (i) determine the testicular gene expression networks underlying these observations, and (ii) define DIO3 role in regulating TH homeostasis in this tissue. Using RNA-sequencing (n=4), we profiled gene expression in the testis of postnatal day 5 Dio3-/-mice and identified 1607 genes differentially expressed (DEGs) by more than 1.5-fold (q<0.05). The altered expression of 23 genes, some of them involved in steroidogenesis and in the formation of the testis-blood barrier, was confirmed by qPCR. DAVID-based gene ontology analysis revealed a profound functional dichotomy between up- and down-regulated genes. Genes involved in cell proliferation, cell cycle, and chromosome and chromatin organization were markedly overrepresented among 637 down-regulated genes (-Log P value range: 20-56), while genes involved in glycoprotein processing and cell membrane functions were overrepresented among 970 up-regulated genes (-Log P value range: 14-42). DEGs whose expression have also been found altered in a published mouse model of SC-specific androgen receptor deficiency showed an significant inverse correlation in their expression changes between the two models. To understand the cellular roles of DIO3 in the developing testis, we genetically inactivated DIO3 in vivoin Sertoli cells (SCs) using a mouse model carrying a floxed Dio3(Dio3f/f) and a Amh-credriver). This resulted in effective Dio3gene recombination in neonatal testis, but intact levels of DIO3 enzymatic activity, suggesting that DIO3 is not appreciably expressed in SCs. In contrast, DIO3 inactivation in spermatogonia (using Dio3f/fmice carrying a Stra8-cretransgene) resulted in >90% inactivation of DIO3 activity in the postnatal testis, demonstrating that testicular DIO3 is predominantly active in spermatogonia. Furthermore,Dio3f/f/Stra8-cremice manifested significant reductions in testis size and SC number at weaning, although these reductions were moderate compared to those observed in Dio3-/-mice. The testes of Dio3f/f/Stra8-creneonates also exhibited marked alterations in the expression of identified TH-regulated genes specific to SC and Leydig cells. Our findings (i) identify new TH-regulated genes in the developing testis that suggest a critical role for TH in arresting cell proliferation and promoting cell differentiation in this tissue, and (ii) indicate that spermatogonial DIO3 influences TH action in the principal cell types in the tetis, defining a novel cross-talk mechanism between germ and somatic cells in the testis that involves the regulation of TH signaling.