Transcriptional silencing of estrogen receptor alpha (ER alpha) expression is an important etiology contributing to, the letrozole-resistance in ER alpha-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ER alpha expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ER alpha expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERa level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ER alpha-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. (C) 2017 Elsevier Inc. All rights reserved.