The constitutive activation of the Stat3 oncogene product and mutation of the p53 tumor suppressor are both frequently detected in human breast cancer. We sought to determine whether there is functional regulation of Stat3 by wild-type (wt) p53. We demonstrate that expression of wt p53, but not mutant p53, significantly diminished phosphorylation of Stat3, reduced Stat3 DNA binding activity, and inhibited Stat3-dependent transcriptional activity in breast cancer cells expressing constitutively active Stat3. Expression of wt p53 did not cause a reduction in the phosphorylation of three unrelated protein kinases in other signal transduction pathways, AKT, extracellular signal-regulated kinase (ERK)1, and ERK2 or a reduction of phosphorylation of epidermal growth factor receptor. Furthermore, the expression of the p53 downstream target, p21(WAF-1), did not have an inhibitory effect on Stat3 phosphorylation. Wt p53 also induced significant apoptosis in breast cancer cell lines that express constitutively active Stat3. Interestingly, the p53-dependent apoptosis occurred in the presence of high levels of phosphorylated AKT and ERK1/2. Therefore, these findings demonstrate a novel p53-dependent cellular process that regulates Stat3 phosphorylation and activity.