Human and animal studies suggest an interaction between the Pro12Ala polymorphism of PPARG and dietary fat. In this randomized crossover clinical trial, we investigated whether subjects with the Pro12Pro and Ala12Ala genotypes of PPARG respond differently to a diet supplemented with high saturated (SAFA) or polyunsaturated fatty acid (PUFA).We recruited non-diabetic men from a population-based METSIM study (including 10,197 men) to obtain men with the Ala12Ala and the Pro12Pro genotypes matched for age and body mass index. Seventeen men with the Pro12Pro genotype and 14 with the Ala12Ala genotype were randomized to both a PUFA diet and a SAFA diet for 8 weeks in a crossover setting. Serum lipids and adipose tissue mRNA expression were measured during the diet intervention. At baseline, subjects with the Ala12Ala genotype had higher levels of HDL cholesterol and lower levels of LDL cholesterol, total triglycerides, and apolipoprotein B compared to those subjects with the Pro12Pro genotype (P < 0.05, FDR < 0.1). The Ala12Ala genotype also associated with higher mRNA expression of PPARG2, LPIN1, and SREBP-1c compared to participants with the Pro12Pro genotype (FDR < 0.001). On the other hand, PUFA diet resulted in lower levels of fasting glucose, total cholesterol, total triglycerides, and apolipoprotein B (P < 0.05, FDR < 0.1) but did not affect PPARG2 mRNA expression in adipose tissue. We conclude that individuals with the Pro12Pro genotype, with higher triglyceride levels at baseline, are more likely to benefit from the PUFA diet. However, the beneficial effects of dietary PUFA and the Ala12Ala genotype of PPARG on serum lipids are mediated through divergent mechanisms.