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Calcineurin is an essential calcium‐activated serine/threonine phosphatase. The six NMR‐observable methionine methyl groups in the catalytic domain of human calcineurin Aα (CNA) were assigned and used as reporters of the presence of potential cis‐trans isomers in solution. Proline 84 is found in the cis conformation in most calcineurin X‐ray structures, and proline 309, which is part of a highly conserved motif in phosphoprotein phosphatases, was modeled with a cis peptide bond in one of the two molecules present in the asymmetric unit of CNA. We mutated each of the two prolines to alanine to force the trans conformation. Solution NMR shows that the P84A CNA mutant exists in two forms, compatible with cis‐trans isomers, while the P309A mutant is predominantly in the trans conformation., The work was partially supported by funds from the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO) (BIO2016‐78006R, to MP; BFU2015‐71092‐P to IF; SAF2015‐66365 and RTC‐2015‐3386‐1, to MP‐R; Units of Excellence María de Maeztu awards to IF (MDM‐2014‐0435) and to JCP (MDM‐2017–076). Some of the funds were cofinanced with the European Fund for Regional Development (FEDER). We acknowledge additional support from Generalitat de Catalunya (2014‐SGR‐541 and 2017‐SGR‐191). A.B. was funded by the RTC‐2015‐3381‐1 grant. S.C. was a recipient of a short‐term fellowship from the Iranian Government.