Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer’s disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.