Fatty acid synthase (FAS)-dependent endogenous fatty acid synthetic activity is abnormally elevated in a biologically-aggressive subset of breast carcinomas. Remarkably, tumor-associated FAS hyperactivity represents a novel target for anti-metabolic therapy because pharmacological inhibitors of FAS are selectively cytotoxic for tumor cells, triggering their apoptotic cell death. Since the p53 tumor-suppressor protein (TP53) is thought to play a novel role in cellular responses of a variety of non-genotoxic metabolic stresses, we characterized the involvement of TP53 in the response of breast cancer cells to FAS inhibition. MCF-7 breast cancer cells were selected for study because they have an intact TP53 pathway and undergo little apoptosis following FAS blockade. Two chemically distinct inhibitors of FAS (the natural mycotoxin cerulenin and the novel small-molecule inhibitor C75) were studied in parallel to provide a broad picture of consequences suffered by the loss of FAS function on TP53 signaling. Treatment with either cerulenin or C75 induced TP53 protein accumulation at 24 h in MCF-7 cells. To determine whether the up-regulation of TP53 following exposure to cerulenin or C75 was solely due to inhibition of endogenous fatty acid metabolism, we first evaluated the cytotoxic response to chemical FAS blockers on MCF-7 cells in which FAS gene expression was previously silenced by using the highly sequence-specific mechanism of RNA interference. MCF-7 cells became insensitive to C75-induced cytotoxicity when the expression of FAS was specifically suppressed by targeted knock-down with small interfering RNA (siRNA), whereas they partially retained their sensitivity to cerulenin. These results demonstrate that C75-induced cytotoxic damage to breast cancer cells is closely dependent on its ability to inhibit FAS-catalyzed endogenous fatty acid biogenesis, thus ruling out a significant direct effect of C75 on DNA. To determine the functional role of TP53 on breast cancer cell survival after FAS blockade, we evaluated FAS inhibitor-mediated apoptosis in MCF-7 cells transiently transfected with a pool of sequence-specific double-stranded RNA oligonucleotides targeting TP53 gene. In these conditions, TP53 protein levels were unchanged during the period of FAS-inhibitor exposure. Remarkably, siRNA-induced silencing of TP53 gene expression did result in a dramatic increase (approximately 300%) in apoptotic cell death following exposure to C75. Strikingly, there was no apparent relationship between the TP53 mutational status and sensitivity to chemical FAS inhibitor in a panel of human breast cancer cell lines. However, the degree of TP53 mRNA expression was predictive of sensitivity to C75-induced cytotoxicity, with low-TP53 mRNA expressing breast cancer cells showing hypersensitivity to FAS blockade. These findings strongly suggest that: a) TP53 is a novel molecular sensor of energy imbalance after the perturbation of endogenous fatty acid metabolism in breast cancer cells; b) TP53 function closely influences the decision between apoptosis and growth arrest following FAS blockade; and c) pharmacological inhibitors of FAS activity may be clinically useful against breast carcinomas exhibiting mutation or aberrant expression of TP53.