Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and neurological complications in young children. Although the underlying mechanisms remain obscure, impaired or aberrant immunity is thought to play a role. In infected cells, EV71 suppresses type I interferon responses mediated by retinoid acid-inducible gene I (RIG-I). This involves the EV71 3C protein, which disrupts the formation of a functional RIG-I complex. In the present study, we report that EV71 inhibits the induction of innate immunity by Toll-like receptor 3 (TLR3) via a distinct mechanism. In HeLa cells stimulated with poly(I · C), EV71 inactivates interferon regulatory factor 3 and drastically suppresses interferon-stimulated gene expression. Notably, EV71 specifically downregulates a TRIF, TIR domain-containing adaptor inducing beta interferon (IFN-β). When expressed alone in mammalian cells, EV71 3C is capable of exhibiting these activities. EV71 3C associates with and induces TRIF cleavage in the presence of Z-VAD-FMK, a caspase inhibitor. TRIF cleavage depends on its amino acid pair Q312-S313, which resembles a proteolytic site of picornavirus 3C proteases. Further, site-specific 3C mutants with a defective protease activity bind TRIF but fail to mediate TRIF cleavage. Consequently, these 3C mutants are unable to inhibit NF-κB and IFN-β promoter activation. TRIF cleavage mediated by EV71 may be a mechanism to impair type I IFN production in response to Toll-like receptor 3 (TLR3) activation.