Abstract Body: Disruption of mitochondrial homeostasis is a root cause of cardiomyopathy and heart failure, but the upstream mechanisms regulating this process remain incompletely understood. Here we report neddylation as a novel post-translational mechanism safeguarding mitochondria integrity in the heart. Neddylation is a reversible post-translational modification that involves the covalent attachment of the ubiquitin-like protein NEDD8 to substrate proteins via NEDD8-specific E1-E2-E3 enzymes. In this study, we targeted NEDD8-activating enzyme 1 (NAE1)- a key subunit of the neddylation E1 enzyme- in adult mouse hearts using low-dose tamoxifen (TAM). Loss of NAE1 led to overt heart failure by 10 weeks post-TAM and ultimately premature death. Transcriptomic analysis of 4-week post-TAM hearts identified perturbations in cardiac metabolism and mitochondrial bioenergetics among other pathways in the mutant hearts, which are validated by biochemical analyses in isolated NAE1-deficient adult cardiomyocytes (CMs) in vitro and hearts in vivo. The deficits in energy are attributed to disrupted mitochondrial dynamics and turnover, as evidenced by massive strikingly elongated mitochondria, the accumulation of mitophagic vesicles and alterations in key regulators such as MFN2, DRP1, PINK1, LC3-II and P62. To understand the contribution of disrupted mitochondrial dynamics, we generated double knockouts (KOs) of NAE1 with either MFN2 (a key mitofusion protein) or DRP1 (a critical mito-fission protein). Intriguingly, MFN2-KO had minimal impact on disease progression of NAE1-deficient mice, while DRP1-KO exhibited accelerated cardiac dysfunction compared to NAE1- or DRP1- single-KO hearts, indicating the pathogenic role of mitochondrial hyperfusion in NAE1-deficient hearts. Furthermore, we found that inhibition of neddylation inactivates cullin family proteins, key scaffold proteins of multi-subunit cullin-RING ubiquitin ligases, thereby inhibiting mitochondrial ubiquitination and consequently their turnover. Taken together, these results uncover a previously unrecognized role of neddylation in balancing mitochondrial dynamics and surveilling mitochondria integrity, highlighting its critical role in sustaining adult heart function.