Abstract Body: Background: ATP-dependent Lonp1 is essential for mitochondrial proteostasis by degrading abnormal proteins within the mitochondrial matrix, however, whether Lonp1 has a protease-independent role in the heart remains unclear. Hypothesis: We hypothesize that Lonp1, protease independently regulates mitochondrial import, assembly of inner membrane proteins required for crisate integrity and thus electron transport chain assembly and ATP production in the heart Methods: We employed Cre-control and cardiac-specific Lonp1 knock-out (Lonp1^cKO) mice and assessed their gross morphology, cardiac structure by histology (Trichrome and WGA), mitochondrial and cristae integrity by Transmission electron microscopy (TEM). We further analyzed mitochondrial-specific proteomic changes, genes (RT-PCR) and protein expression by western blot. Cardiac and mitochondrial function evaluated by echocardiography, oxygen consumption rate (OCR), coupling efficiency and electron transport chain (ETC) complex activities (I-V) respectively. Statistical significance between groups was determined using the student’s t-test, with a threshold of p<0.05. Results: We found that compared to Cre control, Lonp1 cKO mice show reduced body weight (7.02±0.16 gm vs. control (11.41±0.25 gm), p<0.0001, n=24), increased heart weight (0.080±0.002 gm/cm vs. control (0.047±0.0014 gm/cm), p<0.001, n=24) resulting in dilated cardiomyopathy with no mice survived beyond day 21 (P21). The Lonp1cKO mice cardiac mitochondria showed disrupted inner mitochondrial membrane accompanied with electron-dense aggregation, with reduced cristae number (15.60±1.5 vs. cre control (37.7±22.1), p<0.0001, n=6) and its area (0.099±0.062 vs. cre control (0.727±0.62), p<0.0001, n=6). However, Lonp1cKO hearts showed a significant increase in mitochondrial fragmentation (112.4±7.02% vs. cre control (72.4±40), p<0.0005, n=6). Mitochondrial specific proteomics, RT-PCR and western blot analysis revealed significant reduction in most inner mitochondrial membrane (IMM) and ETC complex proteins in Lonp1cKO mice compared to cre control. Histological analysis revealed severe ventricular dilation, increased cardiac fibrosis, and WGA staining revealed loss of cardiomyocytes in Lonp1 cKO mice compared to cre-control. Conclusion: Our findings suggest that Lonp1 plays a pivotal role in inner mitochondrial membrane integrity, possibly by a protease-independent role of importing, assembling IMM proteins for efficient energy production in the heart.