Endurance exercise is an important way to resist and treat high-fat diet (HFD)-induced lipotoxic cardiomyopathy, but the underlying molecular mechanisms are poorly understood. Here, we used Drosophila to identify whether cardiac Nmnat/NAD+/SIR2 pathway activation mediates endurance exercise-induced resistance to lipotoxic cardiomyopathy. The results showed that endurance exercise activated the cardiac Nmnat/NAD+/SIR2/FOXO pathway and the Nmnat/NAD+/SIR2/PGC-1α pathway, including up-regulating cardiac Nmnat, SIR2, FOXO and PGC-1α expression, superoxide dismutase (SOD) activity and NAD+ levels, and it prevented HFD-induced or cardiac Nmnat knockdown-induced cardiac lipid accumulation, malondialdehyde (MDA) content and fibrillation increase, and fractional shortening decrease. Cardiac Nmnat overexpression also activated heart Nmnat/NAD+/SIR2 pathways and resisted HFD-induced cardiac malfunction, but it could not protect against HFD-induced lifespan reduction and locomotor impairment. Exercise improved lifespan and mobility in cardiac Nmnat knockdown flies. Therefore, the current results confirm that cardiac Nmnat/NAD+/SIR2 pathways are important antagonists of HFD-induced lipotoxic cardiomyopathy. Cardiac Nmnat/NAD+/SIR2 pathway activation is an important underlying molecular mechanism by which endurance exercise and cardiac Nmnat overexpression give protection against lipotoxic cardiomyopathy in Drosophila.