Although tyrosine kinase inhibitors have revolutionized treatment for chronic myeloid leukemia (CML), some patients exhibit drug resistance and poor clinical outcomes. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR). TR is an antipsychotic drug that has been investigated for its anticancer potential in a Philadelphia-positive human CML model. Our data show that TR selectively induced apoptosis in K562 cells compared with normal blood cells, which was mediated by Ca2+ and mitochondrial permeabilization. TR-induced cell death was accompanied by changes in Bcl-2 proteins and endoplasmic reticulum stress. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells and presented an antitumor effect in vivo. These findings emphasize the high pharmacological potential of TR as an adjuvant in CML chemotherapy.