Previously we have shown that both Rac1 and c-Jun N-terminal kinase (JNK1/2) are key proapoptotic molecules in TNF-/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells (IEC-6 cells), whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The current studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF--induced apoptosis. In this study, we showed that TNF-/CHX-induced ROS production and hydrogen peroxide (H₂O₂)-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF--induced ROS production. The anti-oxidant, N-acetylcysteine, or rotenone, the mitochondrial electron transport chain (ETC) inhibitor, attenuated mitochondrial ROS production and apoptosis. Rotenone also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF--induced mitochondrial ROS production. Moreover, TNF--induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and N-acetylcysteine. In addition, DPI inhibited TNF--induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1- or cytosolic ROS- dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus, these results indicate that TNF--induced apoptosis requires Rac1-dependent-ROS production in intestinal epithelial cells.