A recently studied endoplasmic reticulum (ER) stress regulator, Bax inhibitor-1 (BI-1) plays a regulatory role in mitochondrial Ca(2+) levels. In this study, we identified ER-resident and mitochondria-associated ER membrane (MAM)-resident populations of BI-1. ER stress increased mitochondrial Ca(2+) to a lesser extent in BI-1-overexpressing cells (HT1080/BI-1) than in control cells, most likely as a result of impaired mitochondrial Ca(2+) intake ability and lower basal levels of intra-ER Ca(2+). Moreover, opening of the Ca(2+)-induced mitochondrial permeability transition pore (PTP) and cytochrome c release were regulated by BI-1. In HT1080/BI-1, the basal mitochondrial membrane potential was low and also resistant to Ca(2+) compared with control cells. The activity of the mitochondrial membrane potential-dependent mitochondrial Ca(2+) intake pore, the Ca(2+) uniporter, was reduced in the presence of BI-1. This study also showed that instead of Ca(2+), other cations including K(+) enter the mitochondria of HT1080/BI-1 through mitochondrial Ca(2+)-dependent ion channels, providing a possible mechanism by which mitochondrial Ca(2+) intake is reduced, leading to cell protection. We propose a model in which BI-1-mediated sequential regulation of the mitochondrial Ca(2+) uniporter and Ca(2+)-dependent K(+) channel opening inhibits mitochondrial Ca(2+) intake, thereby inhibiting PTP function and leading to cell protection.