Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.