Abstract: Objective To explore the mechanism of arsenite-induced permeability transition pore (PTP) opening and the role of Ca2+ in As2O3-induced PTP opening. Method The mitochondria were prepared from Wistar rat liver and mitochondrial swelling was assessed spectrophotometrically at 540 nm to evaluate PTP opening. The membrane potential of the mitochondia was measured with fluorescence spectrophotometry. Results PTP opening was induced with 10 μmol/L As2O3 in the presence of 10 μmol/L Ca2+, and the absorbance at 540 nm of the mitochondria did not decrease in response to exclusive treatment with 10 μmol/L As2O3, or to 10 μmol/L As2O3 plus 10 μmol/L Ca2+ treatment with 0.5 mmol/L EGTA pretreatment. Treatment with As2O3 at 0, 5, 10 and 20 μmol/L in the presence of 50, 20, 10 and 5 μmol/L Ca2+, respectively, resulted in decreased absorbance at 540 nm of the mitochondria. Conclusion Ca2+ mediates As2O3-induced PTP opening. As2O3 lowers Ca2+ threshold necessary for eliciting PTP opening and thereby regulates cell apoptosis.