Activation of Toll-like receptors (TLRs) leads to proinflammatory cytokine production which is responsible for activating the innate immune system. Thus, TLR signaling is subject to multilayer regulatory control mechanisms to prevent a protective response from causing injury. In this study, we report that the E3 ubiquitin ligase RNF182 is highly expressed in macrophages and is specifically upregulated by TLR stimuli (TLR4, TLR3, and TLR9 agonists). Knockdown of RNF182 selectively amplifies TLR signaling by promoting the production of proinflammatory cytokines but not type I interferons in macrophages. Mechanistically, RNF182 promotes the degradation of p65 via K48-linked ubiquitination, resulting in the inhibition of TLR-triggered innate immune responses. Our findings elucidate a feedback-negative mechanism for terminating TLR-induced inflammation and maintaining the immunological balance. This article is protected by copyright. All rights reserved.