Double stranded RNA (dsRNA) is a common pathogen-associated molecular pattern that is recognized by intracellular receptors like Toll-like receptor 3 and used by virus-infected cells to activate the immune response. The transport of dsRNA to TLR3 in endosomes is essential for their signaling function. The proteins flotillin1 and 2 which form heterooligomers are markers of lipid rafts but it is unknown whether they contribute to raft formation or endosome internalization. We studied whether flotillin1 is required for the dsRNA-induced signaling in human umbilical vein endothelial cells (HUVECs). Knockdown of flotillin1 by shRNA leads to a decreased internalization of TLR3 agonist polyinosinic-polycytidilic acid rhodamine (poly I:C rohdamine). Similarly, flotillin1 shRNA attenuated the TLR3-mediated induction of the adhesion molecules VCAM and ICAM as well as adhesion of peripheral blood mononuclear cells to HUVECs in response to agonist-stimulation. Importantly, overexpression of flotillin1 in cells exposed to flotillin1 shRNA restored TLR3-dependent VCAM and ICAM expression excluding non-specific effects of the shRNA. Flotillin1 silencing also disrupted the TLR3-TRIF-dependent signaling pathway as demonstrated by a loss of the phosphorylation of the TLR-downstream target interferon regulatory factor-3 (IRF3). Surprisingly, knockdown of flotillin1 diminished the abundance of the lipid raft protein caveolin1. Additionally, caveolin1 depletion decreased also the flotillin1 level and recapitulates the flotillin1-knockdown phenotype. Moreover caveolin1 and flotillin1 interact in intracellular vesicles but not in the plasma membrane. This may suggest that vesicular interaction between flotillin1 and caveolin1 mediates the transport of dsRNA to TLR3 in endosomes and enables TLR3 signaling.