Obesity-driven cardiac lipid accumulation can progress to lipotoxic cardiomyopathy. Soluble epoxide hydrolase (sEH) is the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), which have biological activity of regulating lipid metabolism. The current study explores the unknown role of sEH deficiency in lipotoxic cardiomyopathy and its underlying mechanism. Wild-type and Ephx2 knock out (sEH KO) C57BL/6 J mice were fed with high-fat diet (HFD) for 24 weeks to induce lipotoxic cardiomyopathy animal models. Palmitic acid (PA) was utilized to induce lipotoxicity to cardiomyocytes for in vitro study. We found sEH KO, independent of plasma lipid and blood pressures, significantly attenuated HFD-induced myocardial lipid accumulation and cardiac dysfunction in vivo. HFD-induced lipotoxic cardiomyopathy and dysfunction of adenosine 5'-monophosphate-activated protein kinase-mammalian target of rapamycin complex (AMPK-mTORC) signaling mediated lipid autophagy in heart were restored by sEH KO. In primary neonatal mouse cardiomyocytes, both sEH KO and sEH substrate EETs plus sEH inhibitor AUDA treatments attenuated PA-induced lipid accumulation. These effects were blocked by inhibition of AMPK or autophagy. The outcomes were supported by the results that sEH KO and EETs plus AUDA rescued HFD- and PA-induced impairment of autophagy upstream signaling of AMPK-mTORC, respectively. These findings revealed that sEH deficiency played an important role in attenuating myocardial lipid accumulation and provided new insights into treating lipotoxic cardiomyopathy. Regulation of autophagy via AMPK-mTORC signaling pathway is one of the underlying mechanisms.