Background: Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding. Objective: The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction. Methods: Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out. Results: HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain. Conclusion: Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.