Lipotoxicity cardiomyopathy is the result of excessive accumulation and oxidation of toxic lipids in the heart. It is a major threat to patients with diabetes. Glucagon-like peptide-1 (GLP-1) has aroused considerable interest as a novel therapeutic target for diabetes mellitus because it stimulates insulin secretion. Here, we investigated the effects and mechanisms of the GLP-1 analog exendin-4 and the dipeptidyl peptidase-4 inhibitor saxagliptin on cardiac lipid metabolism in diabetic mice (DM). The increased myocardial lipid accumulation, oxidative stress, apoptosis, and cardiac remodeling and dysfunction induced in DM by low streptozotocin doses and high-fat diets were significantly reversed by exendin-4 and saxagliptin treatments for 8 weeks. We found that exendin-4 inhibited abnormal activation of the (PPAR alpha)-CD36 pathway by stimulating protein kinase A (PKA) but suppressing the Rho-associated protein kinase (ROCK) pathway in DM hearts, palmitic acid (PA)-treated rat h9c2 cardiomyocytes (CMs), and isolated adult mouse CMs. Cardioprotection in DM mediated by exendin-4 was abolished by combination therapy with the PPAR alpha agonist wy-14643 but mimicked by PPAR alpha gene deficiency. Therefore, the PPAR alpha pathway accounted for the effects of exendin-4. This conclusion was confirmed in cardiac-restricted overexpression of PPAR alpha mediated by adeno-associated virus serotype-9 containing a cardiac troponin T promoter. Our results provide the first direct evidence that GLP-1 protects cardiac function by inhibiting the ROCK/PPAR alpha pathway, thereby ameliorating lipotoxicity in diabetic cardiomyopathy.