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.