The present study was conducted to investigate the effect of feeding the different levels of the dietary fat on the expression of genes encoding proteins involving energy metabolism, oxidative phosphorylation, and lipid synthesis including peroxisome proliferator-activated receptor gamma (PPARγ) of laying hens in the intestine. Birds fed diets with 3 levels of fat, that is, low (LF), medium (MF), and high fat (HF) were reared from 22 to 42 wk of age. Jejunum tissue was collected at week 42 for gene expression analysis. Dietary fat content as ether extract, net energy to AME ratio, and CP content of 3 treatment groups were as follows: LF: 25, 0.735, 187 (g/kg, DM); MF: 61, 0.739, 185 (g/kg, DM); HF: 73, 0.752, 181 (g/kg, DM). The BW, fat pad weight (g), fat pad–to–BW ratio (%) was the same for all the treatments (P > 0·05). Birds fed a diet containing HF increased the AME daily intake per metabolic BW (BW0.75) (P < 0.05). The expression of jejunal PPARγ was increased in the birds fed MF than that fed LF (P < 0.05). Dietary fat level did not affect the expression of other genes: protein kinase AMP-activated noncatalytic subunit gamma 2, NADH dehydrogenase subunit 2, succinate dehydrogenase complex flavoprotein subunit A, ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1, cytochrome c oxidase subunit III, ATP synthase subunit alpha, avian adenine nucleotide translocator, and acetyl-CoA carboxylase alpha (P > 0·05). The mitochondrial count per cell showed no difference among the 3 groups with different dietary treatments (P > 0·05). The results suggest that PPARγ may be important to the energy expenditure during nutrient absorption, digestion, and metabolism, and respiratory chain complexes, and other genes involving mitochondrial energy metabolism and lipogenesis may be less responsive to dietary treatment.