BACKGROUND/AIM: Clinical trials have shown efficacy of the anti-HER2 monoclonal antibody trastuzumab in metastatic breast cancer patients. The aim of the present study was to elucidate the mechanisms by which up-regulation of fatty acid synthase (FAS) expression confers resistance to trastuzumab in HER2-positive breast cancers. MATERIALS AND METHODS: The expression of FAS as well as the cytotoxic effects of combinatorial treatment of trastuzumab and juglone was investigated by immunoblotting, BrdU incorporation, TUNEL assay, and soft agar assay. RESULTS: Pin1 enhanced EGF-induced SREBP1c promoter activity, resulting in the induction of FAS expression in BT474 cells. In contrast, juglone, a potent Pin1 inhibitor, significantly enhanced trastuzumab-induced FAS down-regulation and cell death in BT474 cells. Furthermore, trastuzumab, when used in combination with gene silencing or chemical inhibition of Pin1, increased cleaved poly(ADP-ribose) polymerase and DNA fragmentation to increase trastuzumab sensitivity. CONCLUSION: Pin1-mediated FAS overexpression is a major regulator of trastuzumab-resistant breast cancer growth and survival.