Notch signaling plays a crucial role in the development of colon cancer; targeting the Notch pathway may sensitize colon cancers to various adjuvant agents. The focus of our current study is to identify natural compounds that target Notch signaling and that might be beneficial for the prevention and treatment of colon cancer. Withaferin-A (WA) is a bioactive compound derived from Withania somnifera, which inhibits Notch-1 signaling and downregulates prosurvival pathways, such as Akt/NF-κB/Bcl-2, in three colon cancer cell lines (HCT-116, SW-480, and SW-620). In addition, WA downregulated the expression of mammalian target of rapamycin signaling components, pS6K and p4E-BP1, and activated c-Jun-NH₂-kinase–mediated apoptosis in colon cancer cells. We also established the molecular link between Notch/Akt/mammalian target of rapamycin signaling by complementary approaches (i.e., overexpression of Notch-1 or inhibition of Notch-1 by small interfering RNA). Our results suggest that WA inhibits Notch-mediated prosurvival signaling, which facilitates c-Jun-NH₂-kinase–mediated apoptosis in colon cancer cell lines. These results underscore the anticancer activity of WA, which exhibits potential for further development for targeted chemotherapy and/or chemoprevention strategies in the context of colon cancer. Mol Cancer Ther; 9(1); 202–210