Peptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 are potent fusion inhibitors. We have recently demonstrated that the unique M-T hook structure preceding the pocket-binding motif of CHR peptide-based inhibitors can greatly improve their antiviral activity. In this study, we applied the M-T hook structure to optimize sifuvirtide (SFT), a potent CHR-derived inhibitor currently under Phase III clinical trials in China. The peptide MT-SFT was generated by incorporating two M-T hook residues (Met-Thr) into the N-terminus of sifuvirtide. Multiple structural and functional approaches were used to determine the biophysical properties and antiviral activity of MT-SFT. The high-resolution crystal structure of MT-SFT reveals a highly conserved M-T hook conformation. Compared with sifuvirtide, MT-SFT exhibited a significant improvement in the ability to bind to the N-terminal heptad repeat, to block the formation of the six helix bundle and to inhibit HIV-1 Env-mediated cell fusion, viral entry and infection. Importantly, MT-SFT was fully active against sifuvirtide- and enfuvirtide (T20)-resistant HIV-1 variants and displayed a high genetic barrier to developing drug resistance. Our studies have verified that the M-T hook structure offers a general strategy for designing novel HIV-1 fusion inhibitors and provide new insights into viral entry and inhibition.