Rotenone is an inhibitor of the mitochondrial electron transport chain complex I, resulting in the generation of reactive oxygen species (ROS). Rotenone has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism is still not fully understood. Here, rotenone showed a strong growth inhibitory effect against human breast cancer MCF‐7 cells. DNA flow cytometric analysis, chromatin condensation, and poly (ADP‐ribose) polymerase (PARP) cleavage indicated rotenone actively induced apoptosis in MCF‐7 cells. The antiapoptotic protein, Bcl‐2, was decreased, whereas the apoptotic protein, Bax, was increased in a time‐dependent manner in rotenone‐induced apoptosis. Moreover, the treatment of rotenone in MCF‐7 cells caused the activation of c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinases (MAPKs), and the inactivation of extracellular signal‐regulated protein kinase 1/2 (ERK1/2). The pharmacological inhibition of JNK and p38 MAPK revealed significant protection against rotenone‐induced apoptosis. Taken together, these results indicate rotenone may induce apoptosis through ROS and JNK/p38 MAPKs activation in MCF‐7 cells. © 2009 Wiley‐Liss, Inc.