Ataxia telangiectasia and Rad3‐related (ATR) is a serine/threonine‐specific kinase that plays an important role in the maintenance of genomic integrity. In this study, we investigated the role of ATR in cell‐cycle arrest by withaferin A (WA), a cancer preventative steroidal lactone derived from Withania somnifera plant abundant in India and surrounding countries. The WA treatment decreased the viability of MCF‐7, MDA‐MB‐231, and SUM159 cells. Exposure of breast cancer cells to WA also resulted in suppression of protein level as well as phosphorylation of ATR and its downstream effector kinase (checkpoint kinase 1; CHK1). Both transcriptional and posttranscriptional mechanisms were involved in the WA‐mediated downregulation of ATR protein. Downregulation of ATR protein expression resulting from WA exposure was not attenuated by overexpression of manganese superoxide dismutase. In contrast, the overexpression of CHK1 attenuated WA‐mediated G2/M arrest and augmented S10 phosphorylation of histone H3, a marker of mitotic arrest. The protein level of ATR was lowered by about 50% in breast tumors of WA‐treated mouse mammary tumor virus‐neu mice when compared with vehicle‐treated controls but the difference was not significant due to small sample size. WA treatment sensitized MDA‐MB‐231 and SUM159 cells to growth inhibition and apoptosis induction by cisplatin. Cisplatin treatment resulted in increased autophosphorylation of ATR (T1989) and CHK1 (S345) phosphorylation that was markedly suppressed in the presence of WA. These results indicate that WA is an inhibitor of ATR in human breast cancer cells.