We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347–361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.