The envelope glycoprotein (Env) enables HIV-1 cell entry through fusion of host-cell and viral membranes induced by the transmembrane subunit gp41. Antibodies targeting the C-terminal sequence of the membrane-proximal external region (C-MPER) block the fusogenic activity of gp41 and achieve neutralization of divergent HIV-1 strains and isolates. Thus, recreating the structure that generates broadly neutralizing C-MPER antibodies during infection is a major goal in HIV vaccine development. Here, we have reconstituted a peptide termed CpreTM-TMD in a membrane environment. This peptide contains the C-MPER epitope and the minimum TMD residues required for the anchorage of the Env glycoprotein to the viral membrane. In addition, we have used antibody 10E8 variants to gauge the antigenic configuration attained by CpreTM-TMD as a function of the membrane cholesterol content, a functional determinant of the HIV envelope and liposome-based vaccines. Differential binding of the 10E8 variants and the trend of the IgG responses recovered from rabbits immunized with liposome-peptide formulations, suggested that cholesterol may restrict 10E8 accessibility to the C-MPER epitope. Our data ruled out the destabilization of the lipid bilayer architecture in CpreTM-TMD-containing membranes, and pointed to the perturbation of the helical conformation by lipid packing as the cause of the antigenic configuration loss induced by cholesterol. Overall, our results provide additional insights into the structural basis of the Env complex anchoring to membranes, and suggest new approaches to the design of effective immunogens directed against the near pan-neutralizing HIV-1 epitope C-MPER.