Orai1 and STIM1 are critical components of Ca²⁺ release-activated Ca²⁺ (CRAC) channels that mediate store-operated Ca²⁺ entry (SOCE) in immune cells. Although it is known that Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (also called CRAC regulator 2A, EFCAB4B or FLJ33805). We show that CRACR2A interacts directly with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca²⁺ concentrations. Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca²⁺ and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca²⁺ binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.