Intracellular reactive oxygen species (ROS) are essential secondary messengers in many signaling cascades governing innate immunity and cellular functions. TLR3 signaling is crucially involved in antiviral innate and inflammatory responses; however, the roles of ROS in TLR3 signaling remain largely unknown. In this study, we show that TLR3-induced ROS generation is required for the activation of NF-κB, IFN-regulatory factor 3, and STAT1-mediated innate immune responses in macrophages. TLR3 induction led to a rapid increase in ROS generation and a physical association between components of the NADPH oxidase (NOX) enzyme complex (NOX2 and p47(phox)) and TLR3 via a Ca(2+)-c-Src tyrosine kinase-dependent pathway. TLR3-induced ROS generation, NOX2, and p47(phox) were required for the phosphorylation and nuclear translocation of STAT1 and STAT2. TLR3-induced activation of STAT1 contributed to the generation of inflammatory mediators, which was significantly attenuated in NOX2- and p47(phox)-deficient macrophages, suggesting a role for ROS-STAT1 in TLR3-mediated innate immune responses. Collectively, these results provide a novel insight into the crucial role that TLR3-ROS signaling plays in innate immune responses by activating STAT1.