SARS-CoV-2 remains in circulation 5 years after the first cases of COVID-19 were reported, during which time several variants have been selected with mutations accumulating especially in the more accessible S1 subunit of the Spike protein (S). Consequently, current vaccine platforms have been updated to ensure effectiveness against the Omicron XBB.1.5 variant, highlighting the need for ongoing surveillance and updates of the antigens included in the immunization strategies. To overcome this limitation, we analyzed in a SARS-CoV-2-infected human cohort the immunogenicity of the highly conserved membrane-proximal external region (MPER) of the S2 subunit of the Spike, which is conserved across the Orthocoronavirinae subfamily. A portion of the patients, even if weakly, did elicit antibodies against the MPER. Additionally, we characterized its structure in a low-polarity environment and in lipid membranes, as well as showing its fusogenic potential, confirming its active involvement in the viral infection process. Therefore, we report the suitability of the MPER as a target for vaccination. Considering the impact that lipid membranes may have on the structure of this region, we assessed its expression in the membranes of eukaryotic cells. For that, we designed wild-type and modified S2-derived DNA sequences including the MPER. The results obtained support the feasibility of designing vaccines focused on the conserved MPER region.