Abstract Calcineurin (CN) is the receptor for two immunophilin–immunosuppressant complexes, Cyp–CsA and FKBP-FK506. It is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). It is also inhibited by its own auto-inhibitory domain (AID). Loop 7 is a β-hairpin within CNA that makes close contact with bound immunophilin–immunosuppressant complexes and with the AID. To investigate the role of Loop 7 in inhibition, we generated a series of deletion and substitution mutants and examined their inhibition by Cyp–CsA, FKBP-FK506 and an AID peptide. Our results demonstrate that the contacts made by Loop 7 are critical for its role in CN inhibition. Intriguingly, single residue deletions of Val314 and neighboring residues increased inhibition by FKBP-FK506 >6-fold, whereas they reduced Cyp–CsA inhibition >3-fold and abolished inhibition by the AID peptide. Most of the single substitution mutations also decreased Cyp–CsA inhibition. Loop 7 thus plays different roles in the inhibition of CN by the different inhibitors.