Bcl-2/E1B-19K-interacting protein 3 (BNIP3) is a member of the apoptotic B-cell lymphoma-2 family that regulates cell death. Although BNIP3 targeted normally to the mitochondrial outer membrane by its transmembrane domain was originally considered to be essential for its pro-apoptotic activity, accumulating evidence has shown that BNIP3 is localized to endoplasmic reticulum at physiological conditions and that forced expression of BNIP3 can initiate cell death via multiple pathways depending on the subcellular compartment it targets. Targeting BNIP3 to endoplasmic reticulum has been shown to participate in cell death during endoplasmic reticulum stress. However, the molecular events responsible for BNIP3-induced cell death in the endoplasmic reticulum remain poorly understood. In the present study, the transmembrane domain of BNIP3 was replaced with a segment of cytochrome b5 that targets BNIP3 into endoplasmic reticulum, which induced cell death as effectively as its wild-type molecule in the SW480 cell line (colon carcinoma). Furthermore, a pan-caspase inhibitor, z-VAD-fmk, and PD150606, a specific calpain inhibitor, both significantly suppressed the endoplasmic reticulum-targeted BNIP3-induced cell death. These results suggest that endoplasmic reticulum-targeted BNIP3 induced a mixed mode of cell death requiring both caspases and calpains.