Abstract: Based on the conserved amino acid sequences (TMFWALF and HHDIGTH) of ω3 fatty acid desaturase from Chlamydomonas reinhardtii and Chlorella vulgaris (GenBank accession Nos. ABL09485 and BAB78717, respectively), a degenerated pair of primers was designed. A gene was cloned from Myrmecia incisa Reisigl H4301 with these primers using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Its homology with C. reinhardtii ω3 fatty acid desaturase reached 69% in amino acid sequence. The full-length cDNA sequence (GenBank accession No. EU658930) was composed of 2330 bp. It comprised a 107-bp 5′-untranslated region (UTR), a 912-bp 3′-UTR with a typical poly A tail, and a 1311-bp open reading frame (ORF) encoding a 436-amino-acid protein with a putative molecular weight of 49.06 kDa and pI at 7.85. The deduced amino acid sequence was characterized by a membrane-bound desaturase with two hydrophobic regions and three conserved histidine-rich motifs. Compared to the DNA sequence of this gene, its coding region was found to be interrupted by 6 introns with splicing sites well matching the GT-AG rule. Quantitative real-time PCR result showed that during the stress course of nitrogen starvation, the relative transcription of ω3 fatty acid desaturase gene increased (P<0.05) at 4 h possibly due to shock, subsequently declined and did significantly from 12 h on (P<0.05). The transcript level of this gene dropped to minimum at 20 h, where it accounted for 11.6% of that at 0 h, thereafter fluctuated at a low level only 23.2% of that at 0 h (P<0.01). Composition of fatty acid analysis illustrated that arachidonic acid percentage of total fatty acids in M. incisa increased gradually during this stress course. On the contrary, the percentages of ω3 pathway products, such as α-linolenic acid and 16:3ω3, descended significantly (P<0.05) from 40 h or 96 h on. These results suggested that the lower relative transcription of ω3 fatty acid desaturase gene was responsible for the decreased percentages of ω3 pathway products, ensuring the biosynthesis and accumulation of arachidonic acid along the ω6 pathway in M. incisa grown under the condition of nitrogen starvation. In addition, 16:2ω6, linoleic acid and arachidonic acid were possibly the substrate of this enzyme encoded by this cloned gene in this microalga.