Sifuvirtide (SFT) is the second generation inhibitor of HIV-1 gp41 fusion protein, which is under phase III clinical trial for anti-HIV. However, over a period of time virus develops resistance against SFT. To overcome resistance, methionine and threonine (MT) amino acids were implemented in the pocket binding domain of SFT namely MT-Sifuvirtide (MTSFT), which have more helicity; higher melting temperature and stability against resistance-virus. To date, the binding stability and functional role of MT-hook against wild type and mutant form of HIV-1 gp41 fusion protein are not known. In the present study, SFT and MTSFT were individually docked with the wild and V10A/A19I/Q24R mutant form of HIV-1 gp41. Further MD simulation was carried out to understand the stability of the ligand–protein complexes. The free energy values were calculated from the MD trajectories to understand the effect of mutation involved in the binding process. MD results revealed helix to loop type conformational changes in N-heptad repeat (NHR) of gp41 due to mutation. Intermolecular interactions reveal that MTSFT forms more strong interactions with NHR of gp41 than SFT, which is critical for six-helix bundle stabilisation. Hence, the results explained the detailed functional role of MT-hook against gp41 fusion at the molecular level.