Abstract Body: Aberrant cardiac metabolism has long been implicated in heart failure (HF), yet its characterization in humans remains incomplete. To address this, we performed metabolomic and RNA sequencing analyses on 48 non-failing (NF) and 39 dilated cardiomyopathy (DCM) human hearts. Among multiple metabolic alterations, one-carbon metabolism—particularly the methionine (Met) cycle—was significantly impaired, with reduced Met and S-adenosylmethionine (SAM), the universal methyl donor. To test whether low SAM contributes to HF, we generated mice with cardiac-specific deletion of SAM synthase (Mat2a-cKO). Constitutive knockout was largely embryonic lethal, and surviving mice developed severe systolic dysfunction and died within 12 weeks. Adult-onset Mat2a deletion resulted in 50% mortality within 2 weeks, accompanied by marked cardiac dysfunction and dilation. We next investigated mechanisms underlying reduced Met/SAM in DCM hearts. Quantitative mapping of the methionine cycle in neonatal rat ventricular myocytes (NRVMs) and in vivo steady-state isotope tracing revealed that Met is primarily imported, with only 20–30% recycled from homocysteine and protein degradation, suggesting that impaired recycling alone cannot explain low Met/SAM in DCM. Finally, we explored how low SAM might drive HF. In both human DCM and Mat2a-cKO hearts, creatine—a methylation product of SAM—was reduced, while its precursor guanidinoacetate (GAA) accumulated. Contrary to the prevailing view that the heart cannot synthesize creatine, NRVMs generated ~50% of their creatine pool over 48 hours, and in vivo infusions showed that ~80% of newly acquired cardiac creatine is locally synthesized. GAA synthesis was also detected in the heart. Attempts to restore creatine via overexpression of creatine transporter 1 (Crt1) failed to increase creatine or rescue HF. Unexpectedly, overexpression of Crt2—a promiscuous creatine/GAA transporter—dramatically rescued heart failure despite further creatine depletion. In summary, Met and SAM are reduced in human DCM; loss of SAM in murine hearts induces severe HF; most cardiomyocyte Met is imported with limited recycling; and creatine is synthesized locally from SAM but its reduction does not drive HF progression. These findings suggest that a defective methionine cycle contributes to HF and that Crt2 overexpression can rescue cardiac dysfunction through a mechanism that requires further investigation.