The selective and reversible insertion of pseudo-proline (PsiPro) Systems in cyclosporin C (CsC) featuring different C(2) substituents at the oxazolidine ring and its impact on the conformational and biological properties is described. The presence of a 5-membered ring exerts drastic effects upon the backbone conformation of CsC as demonstrated by NMR analysis. For example, the number of conformations, in particular in DMSO-d6, is strongly reduced and a cis 1-2 amide bond is induced when dialkylated at the C(2) position, resulting in a complete loss of the binding capacity to its receptor CypA. The reversibility of PsiPro insertion allows the temporary introduction of conformational constraints representing a new strategy in pro-drug design.