Abnormal prion protein (PrP^Sc) generated from the cellular isoform of PrP (PrP^C) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP^Sc was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP^Sc formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.