Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNA-dependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.