Background <p>Emerging evidence suggests that preventing SARS-CoV-2 from entering and infecting host cells represents an effective strategy to limit viral infection, particularly in the context of its ongoing evolution. In this study, a small peptide fragment derived from major histocompatibility complex class I (MHC class I), designated MH-1, was investigated for its ability to interfere with the early stages of SARS-CoV-2 infection.</p> Methods <p>Molecular docking was used to characterize the interaction between MH-1 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. The inhibitory effect of MH-1 on S protein–ACE2 binding was further evaluated using an ACE2-functionalized electrochemical impedance spectroscopy (EIS) biosensing platform. Antiviral efficacy was assessed using SARS-CoV-2&#xa0;S-pseudotyped lentiviruses and SARS-CoV-2 variants in different human cells. In vivo inhibitory efficacy of MH-1 was assessed in the K18-hACE2 mouse model, followed by lung viral load measurement and histopathological assessment.</p> Results <p>MH-1 peptide interacted with the S-RBD and disrupted S protein-ACE2 binding. MH-1 effectively reduced SARS-CoV-2 infection in cells that expressed different levels of ACE2 and TMPRSS2. Furthermore, MH-1 decreased the infection of SARS-CoV-2 in T lymphocytes that highly express HLA-C but have low levels of ACE2 and TMPRSS2. In animal studies, MH-1 reduced the viral load in the lungs of K18-hACE2 mice and reduced the infiltration of immune cells, including macrophages and T cells, into the lungs. Levels of lung damage and inflammatory cytokines were also reduced by MH-1 and restored to normal.</p> Conclusions <p>These findings identify MH-1 as a promising prophylactic or early-stage intervention that inhibits SARS-CoV-2 infection by interfering with spike-mediated infection of pulmonary and immune cells.</p>