We investigated the effect of the A-IV-2 allele, which encodes a Q360H substitution in apolipoprotein (apo) A-IV, and dietary fat on cholesterol absorption in humans. In three separate studies we compared fractional intestinal cholesterol absorption between groups of subjects heterozygous for the A-IV-2 allele (1/2) and homozygous for the common allele (1/1) receiving high cholesterol ( ϳ 800 mg/day) diets with different fatty acid compositions. All subjects had the apoE 3/3 genotype. There was no difference in cholesterol absorption between the two genotype groups receiving a high saturated fat diet (33% of total energy as fat; 18% saturated, 3% polyunsaturated, 12% monounsaturated) or a low fat diet (22% of total energy as fat; 7% saturated, 7% polyunsaturated, 8% monounsaturated) diet. However, on a high polyunsaturated fat diet (32% of total energy as fat; 7% saturated, 13% polyunsaturated, 12% monounsaturated) mean fractional cholesterol absorption was 56.7% ؎ 1.9 in 1/1 subjects versus 47.5% ؎ 2.1 in 1/2 subjects ( P ‫؍‬ 0.004). A post hoc analysis of the effect of the apoA-IV T347S polymorphism across all diets revealed a Q360H ϫ T347S interaction on cholesterol absorption, and suggested that the A-IV-2 allele lowers cholesterol only in subjects with the 347 T/T genotype. We conclude that a complex interaction between apoA-IV genotype and dietary fatty acid composition modulates fractional intestinal cholesterol absorption in humans.