Na⁺-K⁺-Cl^- cotransporter isoform 1 (NKCC1) and reverse-mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ overload and dysfunction. OGD/REOX caused a doubling of mitochondrial releasable Ca²⁺ (p < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial releasable Ca⁺ was reduced by ~ 42% (p < 0.05). Genetic ablation of NKCC1 also reduced mitochondrial Ca²⁺ (Ca²⁺_m) accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ±3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was significantly attenuated (66 ±10 % of controls, p < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer’s buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in Ca²⁺_i which was accompanied by loss of _m and cytochrome C release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca²⁺_m homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia.