Toll-like receptor 4 (TLR4) signaling at the plasma membrane and in endosomes results in distinct contributions to inflammation and host defence. Current understanding indicates that endocytosis of cell surface-activated TLR4 is required to enable subsequent signaling from endosomes. Contrary to this prevailing model, our data show that endosomal TLR4 signaling is not reliant on cell surface-expressed TLR4 or ligand-induced TLR4 endocytosis. Moreover, previously recognized requirements for the accessory molecule CD14 in TLR4 endocytosis and endosomal signaling are likely attributable to CD14 binding as well as trafficking and transferring lipopolysaccharide (LPS) to TLR4 at different subcellular localizations. TLR4 endocytosis requires the TLR4 intracellular signaling domain, contributions by phospholipase C gamma 2, spleen tyrosine kinase, E1/E2 ubiquitination enzymes, but not canonical TLR signaling adaptors and cascades. Thus, our study identifies independently operating TLR4 signaling modes that control TLR4 endocytosis, pro-inflammatory cell surface-derived, as well as endosomal TLR4 signaling. This revised understanding of how TLR4 functions within cells might be harnessed to selectively amplify or restrict TLR4 activation for the development of adjuvants, vaccines and therapeutics.