Abstract Body: Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. Understanding the molecular mechanism governing CVD has become a major focus in developing new therapeutics. Ubiquitin (Ub) like protein post-translational modifications (PTMs) have been known to play an important role in the normal functioning of the heart. UFM1 (ubiquitin-fold modifier 1) is a novel Ub-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade, named ufmylation. Ufmylation regulates diverse cellular processes and is implicated in various human diseases. However, its importance in the heart is unknown. For the very first time, we have reported dysregulation of ufmylation in dilated cardiomyopathy, hypertrophic human patients, and animal models. It remains unclear whether ufmylation of cardiac proteins is required for heart function and if so, how ufmylation regulates cardiac function. To determine the role of UFM1 modification in cardiomyocytes we have generated cardiac-specific UFM1-knockout (UFM1CKO) mice. The UFM1CKO mice developed dilated cardiomyopathy at resting condition, as indicated by the left ventricle (LV) wall thinning, chamber dilatation, and impaired contractility at 4 months of age, which were further exacerbated by 6 months. It appears that UFM1 modification constrains pathological cardiac remodeling by maintaining ER homeostasis and regulating ER stress response. To test the hypothesis that UFM1 modification, instead of UFM1 itself is required to maintain cardiac function, we replenished UFM1CKO mice with cardiac-specific WT or conjugation-deficient UFM1 via AAV9. By echocardiography, we revealed that, unlike WT UFM1, overexpression of the conjugation-deficient mutant failed to attenuate the cardiac remodeling and dysfunction of the UFM1CKO heart. Interestingly, the conjugation-deficient mutant further exacerbated the cardiac dysfunction of the UFM1CKO mice. Extensive fibrosis and upregulation of ER stress response protein were also noted in conjugation-deficient mutant UFM1 injected UFM1CKO heart. Overall this study indicates that modification of proteins by UFM1 is critical to cardiac function via maintaining ER homeostasis.