After an episode of myocardial ischemia, opening of the mitochondrial permeability transition pore (mPTP), at the onset of reperfusion, is a critical determinant of myocyte death. We investigated the role of the mPTP as a target for cardioprotection in the human heart. We subjected human atrial tissue, harvested from patients undergoing cardiac surgery, to a period of lethal hypoxia and investigated the effect of suppressing mPTP opening at the onset of reoxygenation. We found that suppressing mPTP opening at the onset of reoxygenation with known mPTP inhibitors cyclosporin A (CsA, 0.2 μmol/l) and sanglifehrin A (SfA, 1.0 μmol/l) 1) improved recovery of baseline contractile function from 29.4 ± 2.0% under control conditions to 48.7 ± 2.2% with CsA and 46.1 ± 2.3% with SfA (P < 0.01) and 2) improved cell survival from 62.8 ± 5.3% under hypoxic control conditions to 91.4 ± 4.1% with CsA and 87.2 ± 6.2% with SfA (P < 0.001). Furthermore, with a cell model in which oxidative stress was used to induce mPTP opening in human atrial myocytes, we demonstrated directly that CsA and SfA mediated their cardioprotective effects by inhibiting mPTP opening, as evidenced by an extension in the time required to induce mPTP opening from 116 ± 8 s under control conditions to 189 ± 10 s with CsA and 183 ± 12 s with SfA (P < 0.01). We report that suppressing mPTP opening at the onset of reoxygenation protects human myocardium against lethal hypoxia-reoxygenation injury. This suggests that, in the human heart, the mPTP is a viable target for cardioprotection.