Short peptide-based inhibition of fusion remains an attractive goal in antihuman immunodeficiency virus (HIV) research based on its potential for the development of technically and economically desirable antiviral agents. Herein, we report the use of the dithiol bisalkylation reaction to generate a series of m-xylene thioether-stapled 22-residue α-helical peptides that have been identified as fusion inhibitors targeting HIV-1 glycoprotein 41 (gp41). The peptide sequence is based on the helix-zone binding domain of the gp41 C-terminal heptad repeat region. We found that one of these stapled peptides, named hCS6ERE, showed promising inhibitory potency against HIV-1 Env-mediated cell-cell fusion and viral replication at a level comparable to the clinically used 36-mer peptide T20. Furthermore, combining hCS6ERE with a fusion inhibitor having a different target site, such as HP23, produced synergistic anti-HIV-1 activity. Collectively, our study offers new insight into the design of anti-HIV peptides with short sequences.