Dietary polyunsaturated fat is known to suppress expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis. The sterol regulatory element-binding protein (SREBP) has recently been shown to be involved in this suppression. We previously reported that the first 2.1 kb of the FAS promoter are sufficient for transcriptional induction by a high carbohydrate diet as well as suppression by polyunsaturated fat in transgenic mice. Here, we first examined the DNA sequences responsible for SREBP-mediated suppression of FAS promoter activity by polyunsaturated fatty acids (PUFA) in vivo. Feeding polyunsaturated fat prevented both the low-level activation of the -278 FAS promoter which contains the -150 sterol response element (SRE), as well as the maximal activation of the longer -444 FAS promoter. We observed that ectopic expression of the activated form of SREBP in liver prevented PUFA-mediated suppression of both the endogenous FAS and FAS promoter-reporter transgene expression. We also found that the promoter region required for PUFA suppression in vivo is located between -278 to -131, where SREBP functions. Using HepG2 cells, we further examined the specific FAS promoter elements required for PUFA suppression. We found that the -150 SRE, as well as the -65 E-Box, contribute to PUFA suppression of the FAS promoter, at least in vitro.