The Na⁺/H⁺ exchanger (NHE) inhibitor, cariporide, has a cardioprotective effect in various animal models of myocardial ischemia/reperfusion. Recent studies suggested that cariporide interacts with the mitochondrial Ca²⁺ overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affectsthe the mitochondrial Ca²⁺ overload and MPT. The isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). The mitochondrial Ca²⁺ ([Ca²⁺ ]m) and the mitochondrial membrane potential (m) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 µM) prevented ouabain-induced hypercontracture (from 40 ± 2% to 24 ± 2%, p < 0.05) and significantly attenuated the ouabain-induced [Ca²⁺ ]m overload (from 149 ± 6% to 121 ± 5% of the baseline value, p < 0.05), but did not affect the m. These results indicate that cariporide attenuates the [Ca²⁺ ]m overload without the accompanying depolarization of m. Moreover, cariporide increased the time taken to induce the MPT (from 79 ± 11 s to 137 ± 20 s, p < 0.05) and also attenuated PAO-induced hypercontracture (from 59 ± 3% to 50 ± 4%, p < 0.05). Our data indicate that cariporide attenuates [Ca²⁺ ]m overload and MPT. Thus, these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.