Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors α1 and β1, which are commonly referred to as TRα1 and TRβ1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TRα1 and TRβ1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TRαKO), Thrb knockout (TRβKO), and wild-type (WT) mice. Triiodothyronine (T₃) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRβKO mice versus that in their untreated controls, whereas T₃ had a diminished effect on TRαKO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T₃-treated WT and TRβKO testes. In contrast, T₃ had no effect on these parameters in TRαKO mice. In untreated adult TRαKO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRβKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRβKO mice showed comparable increases following hypothyroidism. Our results show that TRαKO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRβKO mice, but not TRαKO mice, have normal Sertoli cell responsiveness to T₃. Thus, effects of exogenous manipulation of T₃ on neonatal Sertoli cell development are predominately mediated through TRα1.