Glycolysis plays an important role in cell homeostasis and growth through not only producing ATP but also supplying glycolytic intermediates to biosynthetic pathways. However, how glycolysis is regulated during cardiac hypertrophy is unknown. Yes-associated protein 1 (YAP), a major transcriptional cofactor in the Hippo signaling pathway, is known to regulate cell growth and energy metabolism. Here we examined whether YAP regulates glycolysis in cardiomyocytes. Glucose stress test was performed in neonatal rat ventricular myocytes (NRVMs) transduced with adenoviruses harboring LacZ or YAP. Overexpressed YAP significantly increased glycolytic function and metabolic intermediates of the glycolytic pathway and upregulated both mRNA and protein levels of glucose transporter 1 (Glut1), thereby stimulating glycolysis. Knockdown of Tead1 or Hif-1α, but not c-Myc, inhibited the YAP-induced upregulation of Glut1 protein in NRVMs. YAP-S94A mutant, which is unable to interact with TEAD1, failed to upregulate Glut1 and increase glycolysis. ChIP assays revealed that YAP, Tead1, and Hif-1α bind to the Glut1 promotor in cardiomyocytes. These results suggest that YAP promotes cardiomyocyte glycolysis through a YAP-TEAD1-HIF-1α-dependent transcriptional upregulation of the Glut1 promoter activity.