Abstract Body: Background: The adult heart primarily relies on fatty acid oxidation (FAO) while maintaining metabolic flexibility to meet its ATP demands. A high-fat diet, a common feature of the Western diet, promotes obesity and systemic metabolic syndrome. This is associated with increased fatty acid oxidation and reduced metabolic flexibility in the heart, leading to mitochondrial dysfunction and ultimately contributing to diastolic failure. LONP1 is a master regulator of mitochondrial proteostasis; however, its role in the heart under FAO-driven metabolic stress remains unclear. Hypothesis: Lonp1 protects the heart from high-fat diet (HFD)-induced mitochondrial dysfunction and the resulting adverse cardiac alterations. Objective. To determine whether inducible cardiac-specific deletion of Lonp1 (Lonp1-icKO) aggravates HFD–induced metabolic stress and promotes cardiac dysfunction. Methods: Six-week-old inducible control (Cont-icKO) and cardiac-specific Lonp1 knockout (Lonp1-icKO) mice were fed either a 60% high-fat diet (HFD) or 10% low-fat diet (LFD) for four months (N=5–10). A subset of LFD and HFD fed mice also received low-dose STZ (40 mg/kg) to model type 2 diabetes-like metabolic stress. Body weight, cardiac function, glucose tolerance, insulin sensitivity, and mitochondrial respiration were assessed. Data were analyzed by two-way ANOVA with Tukey’s post hoc test (p<0.05). Results: HFD significantly increased body weight and impaired glucose tolerance, confirming the development of obesity and hyperglycemia. Lonp1-icKO HFD mice showed significantly less percent weight gain than controls (males: 196.71±4.3 vs 293.40±30.37; females: 163±22.47 vs 213.95±22.479). Lonp1-icKO mice on HFD also exhibited improved glucose tolerance compared with HFD-fed controls. In contrast, in the HFD+STZ model, no significant differences in body weight and glucose tolerance between the control and Lonp1-icKO groups were observed. Notably, Lonp1-icKO male mice on HFD displayed significantly reduced maximal mitochondrial respiration (pmol/min) (240.670±47.058 vs 713±47.83) compared with controls. Conclusion: Cardiac Lonp1 appears to play a disease-dependent role during metabolic stress. Under diet-induced stress, Lonp1 deletion may promote a protective metabolic adaptation, while its influence seems less pronounced under more severe diabetic conditions.