During ischemia or hypoxia an increase in intracellular cytosolic Ca²⁺ induces deleterious events, but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the Ca²⁺ overload via mitochondrial Na⁺-Ca²⁺ exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial (HCRE) cells (proximal tubular origin), were subjected to MI and changes in cytosolic Ca²⁺ ([Ca²⁺]_i), Na⁺ ([Na⁺]_i) and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5 fold increase in [Ca²⁺]_i, reaching 136.5 ± 15.8 nM in the first 3.45 min. Subsequently [Ca²⁺]_i dropped and stabilized to 62.7 ± 7.3 nM by 30 min. The first phase of the transient increase was La³⁺ sensitive, not influenced by diltiazem, and abolished when mitochondria were de-energized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). The subsequent recovery phase was impaired in a Na⁺ free medium and weakened when the mitochondrial NCX was blocked with 7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Thus Ca²⁺ entry is likely mediated by store-operated Ca²⁺ channels (SOCs) and depends on energized mitochondria, while [Ca²⁺]_i recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca²⁺ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells.