The isotonic substitution of extracellular chloride by gluconate (extracellular Cl⁻-free) has been demonstrated to elicit cardioprotection by attenuating ischaemia/reperfusion-induced elevation of intracellular chloride ion concentration ([Cl⁻]_i). However, the downstream mechanism underlying the cardioprotective effect of extracellular Cl⁻-free is not fully established. Here, it was investigated whether extracellular Cl⁻-free attenuates mitochondrial dysfunction after hypoxia/reoxygenation (H/R) and whether mitochondrial permeability transition pore (mPTP) plays a key role in the extracellular Cl⁻-free cardioprotection. H9c2 cells were incubated with or without Cl⁻-free solution, in which Cl⁻ was replaced with equimolar gluconate, during H/R. The involvement of mPTP was determined with atractyloside (Atr), a specific mPTP opener. The results showed that extracellular Cl⁻-free attenuated H/R-induced the elevation of [Cl⁻]_i, accompanied by increase of cell viability and reduction of lactate dehydrogenase release. Moreover, extracellular Cl⁻-free inhibited mPTP opening, and improved mitochondria function, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Intriguingly, pharmacologically opening of the mPTP with Atr attenuated all the protective effects caused by extracellular Cl⁻-free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. These results indicated that extracellular Cl⁻-free protects mitochondria from H/R injury in H9c2 cells and inhibition of mPTP opening is a crucial step in mediating the cardioprotection of extracellular Cl⁻-free.