Alantolactone, an allergenic sesquiterpene lactone, has recently been found to have significant antitumor effects on malignant tumor cells. Here, we investigated the potential effect of alantolactone on Bcr/Abl+ imatinib-sensitive and -resistant cells. Alantolactone treatment resulted in obvious apoptosis in both imatinib-sensitive and -resistant K562 cells, as shown by the increase in Annexin V-positive cells, caspase-3 activation, poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and mitochondrial membrane potential collapse. Alantolactone significantly inhibited NF-κB-dependent reporter gene activity, decreased the DNA-binding activity of NF-ОκB, and blocked TNF-α-induced IκBα phosphorylation. Of interest, the oncogenic Bcr/Abl fusion protein but not its mRNA levels were quickly reduced upon alantolactone exposure in imatinib-sensitive and -resistant K562 cells. Bcr/Abl knockdown enhanced the apoptosis driven by alantolactone. Bcr/Abl protein reduction could not be reversed by the addition of proteasome or caspase-3 inhibitors. The overexpression of p65 inhibited alantolactone-induced apoptosis, whereas p65 or Bcr/Abl silencing enhanced its apoptotic-inducing effect. Furthermore, Bcr/Abl-transfected 32D cells showed more sensitivity to alantolactone than vector-transfected control cells, and the Bcr/Abl protein was depleted, as observed in K562 cells. Finally, alantolactone-induced apoptosis was also observed in primary CD34+ CML leukemic cells. Collectively, these findings suggest that alantolactone is a promising potent agent to fight against CML cells via the inhibition of the NF-κB signaling pathway and depletion of the Bcr/Abl protein.