Abstract Body: Background: The adult heart predominantly relies on fatty acid oxidation for ATP production while maintaining metabolic flexibility. However, in high fat diet (HFD) induced cardiac impairment, excessive reliance on FAO leads to mitochondrial dysfunction and ultimately heart failure. LONP1 is a highly conserved master regulator of mitochondrial proteostasis; however, its role in HFD-induced cardiac dysfunction remains unknown. Hypothesis: We hypothesize that Lonp1 protects the heart from mitochondrial dysfunction in HFD-induced cardiac alterations. Methods: We subjected wild-type mice to either a 60% HFD or a normal diet for four months to assess changes in Lonp1 expression levels. Next, we used inducible cardiac-specific Lonp1 knockout mice (Lonp1-icKO), placed on either a low-fat diet (LFD) or a 60% HFD (N=5-10) for four months. We monitored the body weight and caloric intake biweekly. We also assessed systolic function using echocardiography. We conducted glucose tolerance tests to determine hyperglycemia. Further, we assessed mitochondrial function using real-time electron transport and oxidative phosphorylation coupling efficiency in isolated mitochondria. Additionally, we examined mitochondrial ultrastructure by transmission electron microscopy (TEM). Results: Our findings revealed a significant induction of Lonp1 mRNA levels in HFD hearts (p<0.01). TEM analysis demonstrated significant lipid accumulation and disrupted mitochondrial cristae. Additionally, HFD significantly increased body weight (p<0.001) and fasting glucose levels (p<0.001), confirming prediabetic hyperglycemia in control mice. However, Lonp1-icKO males exhibited a significantly reduced weight gain compared to controls (p<0.001) despite similar caloric intake. Notably, male Lonp1-icKO mice on LFD showed significantly better glucose tolerance compared to the controls (p<0.001.) Furthermore, Lonp1-icKO on HFD showed significantly reduced maximal respiration compared to control. Conclusion: Lonp1 is induced in response to HFD-induced metabolic stress, potentially serving a compensatory role in maintaining mitochondrial function and oxidative metabolism in a sex-dependent manner. The reduced weight gain observed in Lonp1-icKO males, despite comparable caloric intake, suggests a metabolic adaptation linked to Lonp1 deficiency. These findings provide novel insights into LonP1 as a potential therapeutic target for obesity induced cardiac dysfunction.