HIV entry is mediated by the envelope glycoproteins gp120 and gp41. The gp41 subunit contains several functional domains: the N-terminal heptad repeat (NHR) domains fold a triple stranded coiled-coil forming a meta-stable prefusion intermediate. The C-terminal heptad repeat (CHR) subsequently folds onto the hydrophobic grooves of the NHR coiled-coil to form a stable 6-helix bundle, which juxtaposes the viral and cellular membranes for fusion. A conserved salt bridge between Lys(574) in NHR and Asp(632) in CHR plays an essential role in the formation of the six-helix bundle. A multi-functional peptide inhibitor for anti-HIV derived from the CHR of gp41 has been designed. It bears a cholesterol group (Chol) at the C-terminal through which the inhibitor can anchor in the cell membrane, and carries an isothiocyanate (NCS) group at the side chain of Asp(632) through which the inhibitor can bind to target covalently at Lys(574) in NHR. The dual functionalized peptide (NCS-C34-Chol) shows high antiviral activity in vitro and in vivo. The inhibitor reacts specifically and rapidly to NHR from gp41. In addition, it exhibits better stability under the digestion of the Proteinase K than C34 and T20.