Resistance to anticancer drugs can sometimes be overcome by combination treatment with other therapeutic drugs. Here, we showed that phytosphingosine treatment in combination with arsenic trioxide (AS2O3) enhanced cell death of naturally AS2O3-resistant human myeloid leukemia cells. The combination treatment induced an increase in intracellular reactive oxygen species level, mitochondrial relocalization of Bax, poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cytochrome c release from the mitochondria. N-acetyl-L-Cysteine, a thiol-containing anti-oxidant, completely blocked Bax relocalization, PARP-1 activation, and cytochrome c release. Pretreatment of 3,4- dihydro-5-[4-(1-piperidinyl)butoxyl-1(2H)-isoquinolinone, a PARP-1 inhibitor, or PARP-1/small interfering RNA partially attenuated cytochrome c release, whereas the same treatment did not affect Bax relocalization. The combination treatment induced selective activation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK by treatment of SB203580 or expression of dominant-negative forms of p38 MAPK suppressed the combination treatment -induced Bax relocalization but did not affect PARP-1 activation. In addition, antioxidant N-acetyl-L-cysteine completely blocked p38 MAPK activation. These results indicate that phytosphingosine in combination with AS2O3 induces synergistic apoptosis in AS2O3-resistant leukemia cells through the p38 MAPK-mediated mitochondrial translocation of Bax and the PARP-1 activation, and that p38 MAPK and PARP-1 activations are reactive oxygen species dependent. The molecular mechanism that we elucidated in this study may provide insight into the design of future combination cancer therapies to cells intrinsically less sensitive to AS2O3 treatment.