The peroxisome proliferator-activated receptor γ (PPARγ) is a critical regulator of adipogenesis. PPARγ^+/− mice are resistant to high-fat diet-induced obesity and thus PPARγ may mediate physiological responses to dietary fat in other mammals. The aim of this study was to determine whether the human PPARγ proline to alanine substitution polymorphism (Pro12Ala) modifies the association between dietary fat and adiposity and plasma lipids. Subjects (n=2141) were controls selected for three case–control studies nested within the Nurses' Health Study, a large ongoing prospective cohort study. Associations between intake of total fat, fat subtypes and BMI were different in PPARγ 12Ala variant allele-carriers compared with non-carriers. Among homozygous wild-type Pro/Pro individuals, those in the highest quintile of total fat intake, had significantly higher mean body mass index (BMI) compared with those in the lowest quintile (27.3 versus 25.4 kg/m², respectively; P-trend<0.0001) whereas among 12Ala variant allele-carriers there was no significant trend observed between dietary fat intake and BMI (P-trend=0.99; P-interaction=0.003). In contrast, intake of monounsaturated fat was not associated with BMI among homozygous wild-type women but was inversely associated with BMI among 12Ala variant allele-carriers (mean in lowest quintile=27.6 versus mean in highest quintile=25.5 kg/m²; P-trend=0.006; P-interaction=0.003). The relationship between dietary fat intake and plasma lipid concentrations also differed according to PPARγ genotype. These data suggest that PPARγ genotype is an important factor in physiological responses to dietary fat in humans.