Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10−19 ≤ P ≤ 4.5 × 10−18) and D6D (FADS2) (6.05 × 10−8 ≤ P ≤ 4.20 × 10−7) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10−16). The significance of haplotype association with D5D activity (P = 2.19 × 10−17) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10−28) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10−9) and decreased D6D activity (P = 9.16 × 10−6) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.