A new full-length cDNA (PsD6) putatively encoding a a dagger(6)-desaturase was cloned from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5. PsD6 contained an open reading frame of 1380 bp encoding a protein of 459 amino acids. The deduced amino acid sequence showed high similarity to those of other a dagger(6)-desaturases. A recombinant vector expressing PsD6 (pPIC3.5K-D6) was constructed and transformed into Pichia pastoris GS115. The heterologous expressed PsD6 in P. pastoris desaturated linoleic acid to gamma-linolenic acid but not desaturated alpha-linolenic acid to stearidonic acid. The results indicated that PsD6 was a fatty acid a dagger(6)-desaturase and it had a substrate specificity for linoleic acid.