Phospholamban (PLB) is a critical regulator of Ca²⁺ cycling in heart muscle cells, and its gene expression is markedly down-regulated by T₃. Nonetheless, little is known about the molecular mechanisms of T₃-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TRα1 and TRβ1 subtypes, and exhibits T₃-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TRα1, but not TRβ1, is essential for T₃-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences −156 to +64 displayed robust T₃-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TRα1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitation and in vitro binding assays show that TRα1 directly binds at the PLB core promoter region. Furthermore, addition of T₃ triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T₃-dependent repression of PLB in cardiac myocytes is directly facilitated by TRα1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing.