Apart from being an important macronutrient, dietary fat has recently gained much prominence for its role in regulating gene expression. Polyunsaturated fatty acids (PUFAs) affect gene expression through various mechanisms including, but not limited to, changes in membrane composition, intracellular calcium levels, and eicosanoid production. Furthermore, PUFAs and their various metabolites can act at the level of the nucleus, in conjunction with nuclear receptors and transcription factors, to affect the transcription of a variety of genes. Several of these transcription mediators have been identified and include the nuclear receptors peroxisome proliferator-activated receptor (PPAR), hepatocyte nuclear factor (HNF)-4α, and liver X receptor (LXR) and the transcription factors sterol-regulatory element binding protein (SREBP) and nuclear factor-κB (NFκB). Their interaction with PUFAs has been shown to be critical to the regulation of several key genes of lipid metabolism. Working out the mechanisms by which these interactions and consequent effects occur is proving to be complicated but is invaluable to our understanding of the role that dietary fat can play in disease management and prevention.