We report a biochemical and structural characterization of domain 2 of the non-structural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with Cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to Cyclosporin A (CsA) resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both Cyclophilins. The interaction surface on the Cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.