The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest.