Fatty acid synthase (FASE), a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many human epithelial cancers, rendering it an interesting target for antineoplastic therapy. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have silenced the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. RNAi-mediated down-regulation of FASE expression resulted in a major decrease in the synthesis of triglycerides and phospholipids and induced marked morphological changes, including a reduction in cell volume, a loss of cell-cell contacts, and the formation of spider-like extrusions. Furthermore, silencing of the FASE gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. Importantly and in striking contrast with LNCaP cells, RNAi-mediated inhibition of FASE did not influence growth rate or viability of nonmalignant cultured skin fibroblasts. These data indicate that RNAi opens new avenues toward the study of the role of FASE overexpression in tumor cells and provides an interesting and selective alternative to chemical FASE inhibitors in the development of antineoplastic therapy.