Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis. Display omitted •Observation of dynamic intermediated formed during LPS transfer by TEM•Reconstitution of the entire LPS transfer cascade to TLR4-MD2 on TIRF microscopy•Identification of key charged residues in LBP and CD14 for dynamic LPS transfer•CD14-LPS complex interact with TLR4 LRR13-LRR15 domain for LPS transfer to MD2 Lipopolysaccharide (LPS) of Gram-negative bacteria activates host innate immune responses. Ryu et al. investigates dynamic intermediates in the LBP-CD14-mediated LPS transfer to TLR4-MD2 down to the single-molecule resolution with negative-stain TEM and TIRF analysis, thus identifying the key molecular determinants in LBP, CD14, and TLR4 for efficient LPS transfer.