The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as the main mechanism for Ca2+ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca2+, NCX1.1 activity is also strongly regulated by Ca2+ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca2+ regulation. Nevertheless, the mechanisms by which Ca2+ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+. It was further demonstrated that a mutation of an acidic residue involved in Ca2+ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca2+ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca2+ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca2+ ions that bind to CBD1 are important for regulating exchanger activity.