To express a Delta 6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway. Full-length ORF (1448 bp) of Delta 6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Delta 6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b(5) domain. Delta 6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Delta 6Des-Iso is specific to the omega-3 pathway. For the first time production of GLA and SDA in a prokaryotic system was achieved.