This study was undertaken to investigate the link between dietary fat content and intestinal levels of anorectic AŁacylethanolamines (NAEs), including oleoyleth‐anolamide (OEA), palmitoylethanolamide (PEA), and lino‐leoylethanolamide (LEA). Male rats were fed high‐fat diets (HFDs) with variable percentages of fat [20–45% of total energy (E%)] for 1–7 d;afterward, the jejunums were isolated, and jejunal NAE levels were measured by liquid‐chromatography mass spectrometry. Enzyme activities and mRNA expression levels were measured for two synthesizing enzymes, Λ‐acylphosphatidylethanolamine‐specific phospholipase D (NAPE‐PLD) and glycerophos‐phodiesterase (GDE1), and one degrading enzyme, fatty acid amide hydrolase (FAAH). We found a dose‐response relation between the quantity/percentage of dietary fat, irrespective of the energy density, and the reduction of intestinal levels of OEA, PEA, and LEA. The reductions were present afterldof 45E% HFD. LEA, the major NAE species, was shown to have an anorectic potency slightly less than that of OEA but higher than PEA. Regulation at the enzyme level seems not to explain the changes in NAE levels. The results suggest the presence of a fat sensor, mediating the reduced intestinal NAE levels. The intestinal NAE levels are reduced in a dose‐ and time‐dependent manner in response to dietary fat intake, and this may contribute to the well‐known hyperphagic effect of HFDs.—Diep, T. A., Madsen, A. N., Holst, B., Kristiansen, M. M., Wellner, N., Hansen, S. H., Hansen, H. S. Dietary fat decreases intestinal levels of the anorectic lipids through a fat sensor. FASEB J. 25, 765–774 (2011). www.fasebj.org