Context: Cytokine polymorphisms and dietary fat composition may influence the risk of the metabolic syndrome (MetS). Objective: The objective of the study was to determine the relationship between lymphotoxin-alpha (LTA), TNF-alpha, and IL-6 gene polymorphisms with MetS risk and investigate whether plasma fatty acid composition, a biomarker of dietary fat intake, modulated these associations. Design: Polymorphisms (LTA rs915654, TNF-alpha rs1800629, IL-6 rs1800797), biochemical measurements, and plasma fatty acids were determined in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). Results: LTA rs915654 minor A allele carriers and TNF-alpha rs1800629 major G allele homozygotes had increased MetS risk [odds ratio (OR) 1.37 (confidence interval [CI] 1.12-1.66), P = 0.002 and OR 1.35 (CI 1.08-1.70), P = 0.009] compared with their TT homozygotes and A allele carriers. Possession of the IL-6 rs1800797 GG genotype by the LTA and TNF-alpha risk genotype carriers further increased risk of the MetS [OR 2.10 (CI 1.19-3.71) P = 0.009], fasting hyperglycemia [OR 2.65 (CI 1.12-6.28), P = 0.027], high systolic blood pressure [OR 1.99 (CI 1.07-3.72), P = 0.03], and abdominal obesity [OR 1.52 (CI 1.01-2.28), P = 0.04]. Plasma polyunsaturated to saturated fat ratio exacerbated these effects; subjects in the lowest 50th percentile had even greater risk of the MetS [OR 4.40 (CI 1.55-12.45), P = 0.005], fasting hyperglycemia, high systolic blood pressure, and abdominal obesity (P < 0.05). Conclusions: LTA, TNF-alpha, and IL-6 genotype interactions increased MetS risk, which was further exacerbated by a low plasma polyunsaturated to saturated fat exposure, indicating important modulation of genetic risk by dietary fat exposure.