Thefreshwater microalgaHaematococcus pluvialis accumulates large amounts of fatty acids in response to adverse con-ditions. However, the key fatty acid desaturase genes inH. pluvialis remain unknown. In this study, we cloned and functionally char-acterized aΔ12 fatty acid desaturase gene, and designated it asHpFAD2. The open reading frame ofHpFAD2 consisted of 1137 base pairs and encoded 378 amino acids. The deduced polypeptide showed 70％ identity to other endoplasmic reticulumΔ12 fatty acid desaturases, whereas it had only 44％ identity to plastidΔ12 fatty acid desaturases. The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulumΔ12 fatty acid desaturases. Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2. Two additional fatty acids (C16:2 and C18:2) were detected in the yeast transformants. The results indicatedΔ12 desaturation activity and substrate preference for C18:1 over C16:1. The transcriptional levels ofH. pluvialisHpFAD2 at different growth stages were measured by quantitative polymerase chain reaction (PCR), indicating that theHpFAD2 transcriptional levels were significantly higher in red cells than those in green cells. Our study brings more insight into the fatty acid biosynthetic pathway ofH. pluvialis.