Enterovirus-induced myocardial injury can lead to severe heart failure. To date, little is known about the early innate stress response that contributes to host defense in the heart. Toll-like receptor 3 (TLR3) is important in the initiation of the innate antiviral response. We investigated the involvement of TLR3, which recognizes viral double-stranded RNA, on encephalomyocarditis virus (EMCV) infection. To examine the contribution of TLR3 in protection from EMCV infection, we infected mice deficient in TLR3 with 50 plaque-forming units of EMCV. TLR3-deficient (TLR3(-/-)) mice were more susceptible to EMCV infection and had a significantly higher viral load in the heart compared with TLR3(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TLR3(-/-) than in TLR3(+/+)mice. TLR3(-/-) mice had impaired proinflammatory cytokine and chemokine expression in the heart following EMCV infection. However, the expression of interferon-beta was not impaired in EMCV-infected TLR3(-/-) mice. EMCV infection leads to a TLR3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury.