Parthenolide is the main bioactive component in feverfew, a common used herbal medicine, and has been extensively studied in relation to its anti-cancer properties. However there have been very few in-depth studies of the activities of this compound at the molecular level. Here, we showed that parthenolide increased reactive oxygen species (ROS), induced cell death, activated AMPK and autophagy, and led to M phase cell cycle arrest in breast cancer cells. Removal of ROS inhibited all parthenolide-associated events, such as cell death, AMPK activation, autophagy induction, and cell cycle arrest. Blockade of autophagy relieved cell cycle arrest, whereas inhibition of AMPK activity significantly repressed the induction of both autophagy and cell cycle arrest. These observations clearly showed that parthenolide-driven ROS activated AMPK-autophagy pathway. Furthermore, inhibition of either AMPK or autophagy significantly potentiated parthenolide-induced apoptosis. Therefore, our results show that parthenolide activates both apoptosis pathway and AMPK-autophagy survival pathway through the generation of ROS, and that suppression of AMPK or autophagy can potentially enhance the anti-cancer effect of parthenolide on breast cancer cells.