Bax inhibitor-1 (BI-1) is an evolutionarily conserved membrane protein that protects mammalian cells against endoplasmic reticulum (ER)-stress induced cell death. BI-1 physically interacts and co-localizes with Bcl2 at the ER, where both reduce the Ca²⁺ content. As both proteins have a similar mechanism of cell protection, we asked how they depend on each other. Here, we mapped the Bcl2-binding site on BI-1 using different N- and C-terminal truncated BI-1 proteins. Truncation of the N-terminal cytosolic residues of BI-1 preceding the first transmembrane domain abolished binding to Bcl2. Furthermore, cell-death experiments pointed out that this interaction between Bcl2 and BI-1 was essential for the protective effect of Bcl2 against apoptotic stimuli. We further pinpointed the interaction of Bcl2 with BI-1 to a smaller region by developing a peptide consisting of 18 aa from the BI-1 N-terminus (NP peptide). This peptide dissociated Bcl2 from BI-1 in pull-down experiments, whereas a control peptide (CP), in which we mutated conserved residues, did not. Furthermore, this NP peptide potently induced cell death and potentiated tunicamycin-induced cell death, showing the importance of the Bcl2/BI-1 complex for cell survival. Next, we investigated the effects of this NP peptide on unidirectional ⁴⁵Ca²⁺ flux from permeabilized MEF monolayers, because BI-1 overexpression reduces and BI-1 deficiency increases the ER Ca²⁺ content. The NP peptide inhibited passive ⁴⁵Ca²⁺ leak as well as IP₃-induced ⁴⁵Ca²⁺ release from the ER, indicating that the Bcl2/BI-1 interaction may be involved in the mechanism regulating ER Ca²⁺ content and IP₃-induced ⁴⁵Ca²⁺ release.