Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes disulfide bond formation and assists protein folding, as well as being a structural subunit of microsomal triglyceride transfer protein (MTP) and prolyl 4-hydroxylase (P4HD), and an estrogen and thyroid hormone-binding protein. Previous reports indicate that some endocrine-disrupting chemicals (EDCs) bind to PDI and disturb its functions, and we executed PDI-knockdown to examine the effects of dysfunction of PDI. In this study, the effects of PDI-knockdown were compared among three cell lines: MCF-7, SH-SY5Y and HeLa. PDI-knockdown induced different levels of cytotoxicity among these cell lines. In MCF-7 cells, PDI-knockdown activated apoptotic signaling, causing cytochrome c release from mitochondria and activation of caspase-9, caspase-6, caspase-7 and poly[ADP-ribose]polymerase-1, and the cytotoxicity induced by PDI-knockdown was suppressed by a pan-caspase inhibitor, z-VAD-fmk. These data suggest that cell death induced by PDI-knockdown is caspase-dependent apoptosis in MCF-7 cells.