The study reveals that Toll-like receptor 4 (TLR4) activates two distinct signaling pathways—TIRAP-MyD88 and TRAM-TRIF—through a process involving endocytosis. TLR4 first activates the TIRAP-MyD88 pathway at the plasma membrane, then is internalized and activates the TRAM-TRIF pathway from early endosomes. This sequential activation highlights a unifying theme in innate immune recognition, where type I interferon-inducing receptors signal from intracellular locations. TLR4 is unique among TLRs as it can activate both MyD88-dependent inflammatory responses and TRIF-dependent interferon production. The study shows that TLR4 endocytosis is mediated by dynamin, and that TRAM functions as a sorting adaptor to recruit TRIF to endosomes. TRAM contains a bipartite localization motif that controls its trafficking between the plasma membrane and endosomes. The results suggest that TRAM-TRIF signaling requires TLR4 endocytosis and that TRAF3 localization is critical for type I interferon production. The findings provide a new model for TLR4 signaling, where endocytosis is essential for activating the TRAM-TRIF pathway and inducing type I interferon. The study also highlights the importance of subcellular localization in determining the signaling outcomes of PRRs.The study reveals that Toll-like receptor 4 (TLR4) activates two distinct signaling pathways—TIRAP-MyD88 and TRAM-TRIF—through a process involving endocytosis. TLR4 first activates the TIRAP-MyD88 pathway at the plasma membrane, then is internalized and activates the TRAM-TRIF pathway from early endosomes. This sequential activation highlights a unifying theme in innate immune recognition, where type I interferon-inducing receptors signal from intracellular locations. TLR4 is unique among TLRs as it can activate both MyD88-dependent inflammatory responses and TRIF-dependent interferon production. The study shows that TLR4 endocytosis is mediated by dynamin, and that TRAM functions as a sorting adaptor to recruit TRIF to endosomes. TRAM contains a bipartite localization motif that controls its trafficking between the plasma membrane and endosomes. The results suggest that TRAM-TRIF signaling requires TLR4 endocytosis and that TRAF3 localization is critical for type I interferon production. The findings provide a new model for TLR4 signaling, where endocytosis is essential for activating the TRAM-TRIF pathway and inducing type I interferon. The study also highlights the importance of subcellular localization in determining the signaling outcomes of PRRs.