CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that CD14 enhances double-stranded RNA (dsRNA)-mediated Toll-like receptor 3 (TLR3) activation. Bone marrow-derived macrophages (BMDMs) from CD14-/- mice exhibited impaired responses to polyinosine-polycytidylic acid (pIpC) and reduced production of inflammatory cytokines. CD14-/- mice injected with pIpC also showed impaired cytokine production. When tested with [32P] labeled pIpC small fragments (pIpCsf) that maintain the inflammatory activity of crude pIpC, CD14 directly bound pIpCsf and mediated cellular uptake of pIpCsf. Our data show that TLR3 is intracellular and directly interacts with CD14. Internalized pIpCsf was localized in the lysosomes via the endosomes. In unstimulated cells, neither CD14 nor TLR3 was detected in the lysosomes. However, TLR3 was localized in the lysosomes as was CD14 once the cells took up pIpC. We also observed that internalized pIpCsf colocalized with CD14 and TLR3. Consequently, CD14 mediates pIpC uptake and enhances TLR3 signaling.