Myocarditis is a challenging inflammatory disease of the heart, and better understanding of its pathogenesis is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by CYP (cytochrome P450) enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by sEH (soluble epoxide hydrolase), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and in the murine heart and correlated with the increased expression and activity of sEH after coxsackie B3 virus infection. Administration of a sEH inhibitor prevented coxsackie B3 virus-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated viral infection or improved viral resistance by activating type I IFN (interferon) signaling. At the molecular level, EETs enhanced the interaction between GSK3 beta (glycogen synthase kinase-3 beta) and TBK1 (TANK-binding kinase 1) to promote IFN-beta production. Our findings revealed that EETs and sEH inhibitors prevent the progress of coxsackie B3 virus-induced myocarditis, particularly by promoting viral resistance by increasing IFN production.