The immunosuppressive drug cyclosporin A (CsA) exerts its effects by first binding to cyclophilin (CyP), forming a complex that inhibits calcineurin (CaN). While aiming to develop derivatives with enhanced immunosuppressive activity, we synthesized 2a and 2b, where CsA's MeLeu-4 gemdimethyl group (effector domain) was replaced with sec-butyl. In vitro assays showed 2a had a 4-fold higher affinity for cyclophilin A (CyP-A) than CsA (relative IC50: 0.25 vs 1) but 2-3 times lower immunosuppressive activity in mouse mixed lymphocyte reaction (MLR M) and interleukin-2 reporter gene (IL2 RG) assays. X-ray crystallography of the 2a-CyP-A complex (1.86 Å resolution) revealed the cyclosporin backbone and CyP-A binding interactions were nearly identical to those of the CsA-CyP-A complex, with no significant conformational differences. The improved binding affinity of 2a is likely due to a more favorable equilibrium between its binding and non-binding conformers in aqueous solution. These results highlight that the conformational versatility of cyclosporin in solution is an important factor for designing improved immunosuppressants.