Oleate-Delta(12) desaturase (FAD2) is a key enzyme involved in the conversion of oleic acid (C18:1) into linoleic acid (C18:2). Brassica napus FAD2 gene was targeted for silencing by its RNAi gene under the control of a seed-specific napin promoter. This study aims to identify the transgenic plants, and analyze the level of BnFAD2 transcripts in addition to fatty acid profile in T(3) seeds. As indicated by PCR and southern blotting analysis, a total of six transgenic plants were developed. Analysis performed by RT-PCR revealed the significant down-regulation of BnFAD2 transcripts in developing T(3) seeds, which resulted in 13.90 to 32.20% increase of oleic acid composition in mature T(3) seeds. The data demonstrated that BnFAD2 gene was efficiently down-regulated and mediated by its RNAi gene, and oleic acid composition in transgenic rapeseeds was significantly enhanced.