Withaferin A (WA) is an active steroidal lactone derived from the herbal plant Withania somnifera, which exhibits antitumor activity with reactive oxygen species (ROS) modulating in a variety of cancer models, such as breast cancer, lung cancer and pancreatic cancer. However, to the best of our knowledge, the direct effect and mechanism of WA on CRC cells has not been previously determined. The present study investigated the anti-tumor effects of WA on CRC cells in vitro, and explored the mechanisms of action. The flow cytometry was applied for detecting the accumulation of ROS with the treatment of withaferin A. We performed the flow cytometry and western blot to evaluate the withaferin A induced apoptosis and cell cycle arrest in human colon cancer cells. And to verify the ROS accumulation induced mitochondrial dysfunction after the treatment of withaferin A, fluorescence microscope and western blot were applied. WA exerted a dose-dependent cytotoxic effect on HCT-116 and RKO cells. The effect was associated with ROS-mediated cell cycle arrest and the expression of apoptotic proteins. In addition, WA promoted ROS production and decreased mitochondrial membrane potential accompanying with mitochondrial dysfunction. Taken together, these results strongly indicated that WA directly inhibits cell growth and induces apoptosis in CRC cells through ROS-mediated mitochondrial dysfunction and JNKs pathway, and WA may be a promising potential candidate for therapeutic application of CRC. (C) 2018 Elsevier Inc. All rights reserved.