Abstract Body: Understanding the metabolic and epigenetic mechanisms underlying cardiac aging is essential for developing targeted strategies to preserve or restore heart function in older individuals. We hypothesized that age and sex distinctly influence cardiac metabolic pathways and metabolite cofactors associated with histone and protein acetylation and methylation. To test this, we analyzed heart tissues from female and male C57BL/6JN mice at 4 months (4M, young; n=8), 12M (middle-aged; n=8), and 24M (aged; n=8), kindly provided by the NIA Aged Rodent Tissue Bank. Metabolites were quantified using LC-MS (HPLC-TripleTOF) in heart homogenates. Our untargeted metabolomics analysis revealed significant age-related alterations in cardiac metabolism. Key changes in aged mice included dysregulation of glycolysis, the citric acid cycle, ketone body metabolism, fatty acid synthesis/oxidation, glycolipid metabolism, carnitine biosynthesis, and branched-chain amino acid and glycine-serine-alanine pathways compared to sex-matched young mice. Moreover, aged hearts exhibited disrupted levels of metabolites involved in epigenetic regulation, including intermediates of methylation and acetylation. Notably, sex-specific differences emerged with aging: aged females showed increased tryptophan catabolism and homocysteine degradation, while aged males exhibited relatively higher levels of methyl histidine and β-alanine metabolism. Cardiac pyruvate levels, a key glycolytic product, significantly declined with age (p<0.01), while lactate levels increased (p<0.001) in 24M female and male mice compared to their 4M sex-matched counterparts. Cardiac acetyl-CoA levels were markedly elevated in aged mice of both sexes (female: 24M, 84.69 ± 6.6 vs. 4M, 25.06 ± 2.1; male: 24M, 75.6 ± 4.6 vs. 4M, 20.7 ± 2.3; p<0.0001). Conversely, levels of β-hydroxybutyrate, an anti-aging metabolite and endogenous histone deacetylase inhibitor, were significantly reduced in aged mice (p<0.0001 in males; p<0.01 in females). Additionally, α-ketoglutarate, a tricarboxylic acid cycle intermediate and essential epigenetic cofactor for histone and DNA demethylases, was significantly decreased in aged females (24M, 30.2 ± 3.9 vs. 12M, 50.8 ± 1.7; p<0.01). Our results reveal age- and sex-related alterations in cardiac metabolic pathways and metabolite cofactors of epigenetic enzymes, characterized by an increase in epigenetically modified intermediate metabolites with aging.