Members of the ALMT (Al-Activated Malate Transporter) and MATE (Multidrug And Toxin Efflux) families confer plant aluminum resistance on acid soils by mediating organic acid anion efflux, thereby immobilizing toxic aluminum (Al3+) ions in the rhizosphere. Although similar in function, ALMT and MATE structure and regulation are remarkably different: while ALMT transport activity in heterologous systems is dependent on and enhanced by extracellular Al3+, the MATE transporter mediates constitutive and Al3+-insensitive transport, suggesting that in planta the MATE transport activity is modulated by additional cellular mechanisms associated with an upstream Al3+ signaling cascade. Functional (i.e. electrophysiological) approaches allowed us to screen for and identify the calcineurin B-like (CBL5)/protein kinase (CIPK2) complex as a modulator of AtMATE1 activity. Bimolecular fluorescence complementation analysis in Xenopus oocytes and in planta validated the specificity of the protein-protein interactions among CBL5, CIPK2 and AtMATE1, and suggests a mechanism by which the CBL5/CIPK2 complex alters the trafficking of AtMATE1 into and out of the plasma membrane. We identify CBL5-CIPK2 as part of a calcium-regulated pathway involved in aluminum resistance responses, by which phosphorylation of the downstream target protein (AtMATE1) limits unnecessary carbon loss via unregulated citrate exudation, thereby regulating the abiotic stress tolerance response in a temporal and spatial manner.