Binding of Na+ and Ca2+ ions to the large cytosolic loop of the Na+/Ca2+ exchanger (NCX) regulates its ion transport across the plasma membrane. We determined the solution structures of two Ca2+-binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD) form the regulatory exchanger loop. CBD1 and CBD2 constitute a novel Ca2+-binding motif and are very similar in the Ca2+-bound state. Strikingly, in the absence of Ca2+ the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a sevenfold higher affinity for Ca2+ this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.