Rationale: Heart failure with preserved ejection fraction (HFpEF) is a mortal clinical syndrome without effective therapies. We recently demonstrated in mice that a combination of metabolic and hypertensive stress recapitulates key features of human HFpEF. Objective: Using this novel preclinical HFpEF model, we set out to define and manipulate metabolic dysregulations occurring in HFpEF myocardium. Methods and Results: We observed impairment in mitochondrial fatty acid oxidation associated with hyperacetylation of key enzymes in the pathway. Downregulation of sirtuin 3 and deficiency of NAD ⁺ secondary to an impaired NAD ⁺ salvage pathway contribute to this mitochondrial protein hyperacetylation. Impaired expression of genes involved in NAD ⁺ biosynthesis was confirmed in cardiac tissue from patients with HFpEF. Supplementing HFpEF mice with nicotinamide riboside or a direct activator of NAD ⁺ biosynthesis led to improvement in mitochondrial function and amelioration of the HFpEF phenotype. Conclusions: Collectively, these studies demonstrate that HFpEF is associated with myocardial mitochondrial dysfunction and unveil NAD ⁺ repletion as a promising therapeutic approach in the syndrome.