There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation. J. Cell. Physiol. 209: 153–161, 2006. © 2006 Wiley-Liss, Inc.