Various post transcriptional gene silencing strategies have been developed and exploited to study gene function or engineer disease resistance. The recently developed artificial microRNA strategy is an alternative method of effectively silencing target genes. The Δ12-desaturase (FAD2), Fatty acid elongase (FAE1), and Fatty acyl-ACP thioesterase B (FATB) were targeted with amiR159b-based constructs in Arabidopsisthaliana to evaluate changes in oil composition when expressed with the seed-specific Brassica napus truncated napin (FP1) promoter. Fatty acid profiles from transgenic homozygous seeds reveal that the targeted genes were silenced. The down-regulation of the AtFAD-2 gene substantially increased oleic acid from the normal levels of ∼15% to as high as 63.3 and reduced total PUFA content (18:2(Δ9,12) + 18:3(Δ9,12,15) + 20:2(Δ11,14) + 20:3(Δ11,14,17)) from 46.8 to 4.8%. Δ12-desaturase activity was reduced to levels as low as those in the null fad-2-1 and fad-2-2 mutants. Silencing of the FAE1 gene resulted in the reduction of eicosenoic acid (20:1(Δ11)) to 1.9 from 15.4% and silencing of FATB resulted in the reduction of palmitic acid (16:0) to 4.4% from 8.0%. Reduction in FATB activity is comparable with a FATB knock-out mutant. These results demonstrate for the first time amiR159b constructs targeted against three endogenous seed-expressed genes are clearly able to down-regulate and generate genotypic changes that are inherited stably over three generations.