The Na⁺-Ca²⁺ exchanger plays a central role in cardiac contractility by maintaining Ca²⁺ homeostasis. Two Ca²⁺-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca²⁺ binding to these regulatory domains activates the transport of Ca²⁺ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca²⁺ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca²⁺-bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca²⁺ ions in primary and secondary Ca²⁺ sites. In the absence of Ca²⁺, Lys⁵⁸⁵ stabilizes the structure by coordinating two acidic residues (Asp⁵⁵² and Glu⁶⁴⁸), one from each of the Ca²⁺-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca²⁺ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca²⁺ site completely remove Ca²⁺ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na⁺-Ca²⁺ exchanger.