Tripartite motif (TRIM)38 is an E3 ubiquitin ligase that was reported to regulate signaling ininnate immuneand inflammatory responsesin certain cell lines. In this study, we show that Trim38 deficiency markedly increasedTLR3- and TLR4-mediated induction of type I IFNs and proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, inimmunecells and in vivo.Trim38deficiency also caused the mice to be more susceptible to death triggered by polyinosinic-polycytidylic acid, LPS, and Salmonella typhimurium. Mechanistically,TRIM38catalyzed K48-linked polyubiquitination of theTLR3/4 adapter protein TIR domain-containing adapter-inducing IFN-β at K228 and promoted its proteasomal degradation inimmunecells. Moreover,Trim38was highly induced by type I IFNs, which thennegativelyregulated TNF-α/IL-1β signaling in IFN-β-primedimmunecells, but not unprimedimmunecells, by mediating degradation of Tab2 in a lysosomal-dependent process. These results suggest that Trim38 negatively regulates TLR3/4-mediated innateimmune and inflammatory responses by two sequential and distinct mechanisms. This study increases our understanding of how the innate immune response is initiated during the early phase of infection to defend against microbial invasion and is efficiently terminated during the late phase to prevent excessive and harmful inflammatory responses.