The HIV‐1 gp41 core, a six‐helix bundle formed between the N‐ and C‐terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core‐specific monoclonal antibody (NC‐1). JCH‐4, a peptide containing HXXNPF motif, effectively inhibited HIV‐1 envelope glycoprotein‐mediated syncytium‐formation. The epitope of JCH‐4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core‐binding sequence and HXXNPF motif‐containing molecules can be used as probes for studying the role of the HIV‐1 gp41 core in membrane fusion process.