Abstract Body: Imbalances in triglyceride (TG) catabolism are closely associated with cardiac lipid deposition and contractile dysfunction, as observed in patients with primary triglyceride deposit cardiomyovasculopathy (TGCV) due to adipose triglyceride lipase (ATGL) deficiency. BSCL2/Seipin is a highly conserved protein in the endoplasmic reticulum that plays a key role in lipid droplet (LD) biogenesis and TG metabolism. We previously reported that deleting BSCL2 in the heart increased myocardial ATGL expression and decreased cardiac steatosis. However, the relationship between BSCL2 and ATGL in the heart still needs further investigation. In this study, we unexpectedly found that both global and cardiac-specific deletion of BSCL2 completely alleviated lethal lipotoxic cardiomyopathy in the genetic TGCV model (Atgl^−/− mice and mice with cardiac-specific deletion of Atgl, referred to as A^cKO) by significantly reducing cardiac TG deposits, eliminating cardiac hypertrophy, and restoring cardiac contractility. Transcriptomic analyses revealed elevated inflammation, oxidative stress, and fibrosis in Atgl^−/− hearts, which were dramatically attenuated by BSCL2 deletion. Conversely, the severely impaired cardiac metabolic functions, muscle contraction, and ion transport in Atgl^−/− hearts were remarkably improved by BSCL2 deletion. Notably, the markedly downregulated Pgc1/Ppara and fatty acid oxidation genes in Atgl-deficient hearts were restored by BSCL2 deletion. Further deletion of Pparα reverted Atgl^−/−Bscl2^−/− hearts to lethal lipotoxic cardiomyopathy, indicating its crucial role in the rescue effects mediated by BSCL2 deletion. To identify the pathways that activate PPARα in the absence of ATGL and BSCL2, we conducted quantitative myocardial LD proteomics and discovered distinct LD proteomes characterized by an increased targeting of several neutral lipid hydrolases including hormone sensitive lipase (HSL) to the LDs in hearts with cardiac-specific deletions of both Atgl and Bscl2 compared to A^cKO hearts. These findings suggest cardiac BSCL2 autonomously regulates ATGL-independent neutral lipid metabolism essential for cardiac function by mobilizing the LD proteome. In conclusion, our study reveals novel mechanistic insights into the role of BSCL2 in the LD proteome and offers new therapeutic strategies for treating lethal triglyceride deposit cardiomyopathy.