Store‐operated Ca 2+ channels, which are activated by the emptying of intracellular Ca 2+ stores, provide one major route for Ca 2+ influx. Under physiological conditions of weak intracellular Ca 2+ buffering, the ubiquitous Ca 2+ releasing messenger InsP 3 usually fails to activate any store‐operated Ca 2+ entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca 2+ that has been released by InsP 3, enabling stores to empty sufficiently for store‐operated channels to activate. Here, we report a novel role for mitochondria in regulating store‐operated channels under physiological conditions. Mitochondrial depolarization suppresses store‐operated Ca 2+ influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP 3‐sensitive store depletion, can be distinguished from Ca 2+‐dependent inactivation of the store‐operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca 2+ uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store‐operated influx and raise the possibility of bidirectional Ca 2+‐dependent crosstalk between mitochondria and store‐operated Ca 2+ channels.