Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and up-regulated Cu/Zn superoxide dismutase and heme oxygenase-1 (two antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. Additionally, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a pro-apoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (two apoptogenic proteins), but was independent of caspase cascade activation. While N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG, and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE up-regulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus, modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.