TIR domain-containing adaptor inducing interferon-β (TRIF) is an essential adaptor protein required for innate immune responses mediated by Toll-like receptor (TLR) 3- and TLR4. Here we identify USP19 as a negative regulator of TLR3/4-mediated signaling. USP19 deficiency increases the production of type I interferons (IFN) and proinflammatory cytokines induced by poly(I:C) or LPS in vitro and in vivo. Usp19^-/- mice have more serious inflammation after poly(I:C) or LPS treatment, and are more susceptible to inflammatory damages and death following Salmonella typhimurium infection. Mechanistically, USP19 interacts with TRIF and catalyzes the removal of TRIF K27-linked polyubiquitin moieties, thereby impairing the recruitment of TRIF to TLR3/4. In addition, the RING E3 ubiquitin ligase complex Cullin-3-Rbx1-KCTD10 catalyzes K27-linked polyubiquitination of TRIF at K523, and deficiency of this complex inhibits TLR3/4-mediated innate immune signaling. Our findings thus reveal TRIF K27-linked polyubiquitination and deubiquitination as a critical regulatory mechanism of TLR3/4-mediated innate immune responses.