Background The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-¿B, RXRs, LXRs, FXR, HNF4¿, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. Methodology/Principal Findings Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPAR¿. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPAR¿ agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip® Arrays, fatty acid- and WY14643-induced interactions between PPAR¿ and coregulators were found to be highly similar, although several PPAR¿-coactivator interactions specific for WY14643 were identified. Conclusions/Significance We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPAR¿ and mimic those of synthetic PPAR¿ agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.