Overexpression of the HER2 oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2-overexpressing breast cancer cells, such as SK-BR-3 and BT-474 cells, express fatty acid synthase (FAS) at a higher level than MCF-7 cells or MDA-MB-231 cells, where HER2 is expressed at a moderate or low level. Adenovirus-mediated HER2 expression in MDA-MB-231 cells increased acetyl-CoA carboxylase alpha (ACCα) and FAS protein levels without significant increases of their mRNA. HER2-mediated increases of ACCα and FAS proteins were inhibited by the PI3K inhibitor, LY294002, and the mTOR inhibitor, rapamycin. But sterol regulatory element-binding protein 1 (SREBP1) and ATP citrate lyase (ACL) mRNA and protein levels were not changed by HER2 overexpression, LY294002 or rapamycin. In conclusion, our results suggest that HER2-overexpressing cells lead to increased ACCα and FAS proteins at a translational level via the activation of the mTOR signaling pathway and not through SREBP-1c-mediated transcriptional activation.