Tumor necrosis factor receptor–associated factor 6 (TRAF6) is critical for mediating Toll-like receptor (TLR)–interleukin 1 receptor (IL-1R) signaling and subsequent activation of NF-κB and AP-1, transcriptional activators of innate immunity. Here we show that β-arrestins, a family of multifunctional proteins, directly interacted with TRAF6 after TLR–IL-1R activation. Formation of the β-arrestin–TRAF6 complex prevented autoubiquitination of TRAF6 and activation of NF-κB and AP-1. Endotoxin-treated β-arrestin 2–deficient mice had higher expression of proinflammatory cytokines and were more susceptible to endotoxic shock. Thus, β-arrestins are essential negative regulators of innate immune activation via TLR–IL-1R signaling.