Heart failure is a leading cause of death worldwide. Estrogen-related receptors (ERRs) are a nuclear receptor subfamily that facilitates the transcription of contractile and nucleus-encoded mitochondrial genes in the heart. Impaired expression of these ERR target genes is frequently observed in human heart failure patients. However, the responsible molecular mechanism is not well-understood. Recently, we have shown that PPARα forms a protein complex with Sirt1, which is involved in the downregulation of ERR targets through direct interaction with the ERR response element (ERRE) in the failing heart. Here, we provide additional lines of evidence supporting the pathological involvement of the PPARα/Sirt1 complex in heart failure. Pressure overload-induced left ventricular (LV) hypertrophy was attenuated in mice with heterozygous knockout of either PPARα (PPARα (+/-) ) or Sirt1 (Sirt1 (+/-) ), whereas cardiac-specific PPARα and Sirt1 bigenic mice showed LV hypertrophy accompanied by a high mortality rate even without pressure overload. Microarray analyses indicated that nuclear-encoded mitochondrial genes were largely downregulated and mitochondrial morphological abnormalities were observed in PPARα/Sirt1 bigenic mice. Those downregulated mitochondrial genes frequently harbor the ERRE in the promoter regions. Artificial and physiological PPARα ligands suppressed reporter genes driven by the ERREs. PPARα bound to and recruited Sirt1 to the genomic flanking region of the ERREs in the heart. Pressure overload downregulated many ERR targets, which were partly normalized by PPARα (+/-) and Sirt1 (+/-) mice. These results suggest that PPARα and Sirt1 downregulate ERR target gene expression through direct interaction with the ERRE in the failing heart.