Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine", is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca2+ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca2+ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca2+ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events. Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediatedinflux into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.