Chronic high doses of morphine inhibit the growth of various human cancer cell lines. However, the mechanisms by which such high-dose morphine inhibits cell proliferation and induces cell death are not fully understood. Here we show that c-Jun N-terminal kinase (JNK) plays a pivotal role in high-dose morphine-induced apoptosis of SH-SY5Y cells in a mitochondria-dependent manner. Activation of JNK by morphine led to reactive oxygen species (ROS) generation via the mitochondrial permeability transition pore, because the mPTP inhibitor cyclosporin A significantly inhibited ROS generation. ROS in turn exerted feedback regulation on JNK activation, as shown by the observations that cyclosporin A and the antioxidant N-acetylcysteine significantly inhibited the phosphorylation of JNK induced by morphine. ROS-amplified JNK induced cytochrome c release and caspase-9/3 activation through enhancement of expression of the proapoptotic protein Bim and reduction of expression of the antiapoptotic protein Bcl-2. All of these effects of morphine could be suppressed by the JNK inhibitor SP600125 and N-acetylcysteine. The key role of the JNK pathway in morphine-induced apoptosis was further confirmed by the observation that decreased levels of JNK in cells transfected with specific small interfering RNA resulted in resistance to the proapoptotic effect of morphine. Thus, the present study clearly shows that morphine-induced apoptosis in SH-SY5Y cells involves JNK-dependent activation of the mitochondrial death pathway, and that ROS signaling exerts positive feedback regulation of JNK activity.