The open reading frame of the Delta(6)-desaturase gene was isolated from Mortierella alpina W15 and the gene was cloned into a pPIC3.5K vector. The vector was transformed into Pichia pastoris GS115 and expression was induced with methanol. The Delta(6)-desaturase expressed in P. pastoris GS115 catalyzed the conversion of linoleic acid to gamma-linolenic acid but not the conversion of alpha-linolenic acid to octadecatetraenoic acid. The results indicate that the Delta(6)-desaturase gene from M. alpina W15 has substrate specificity in different organisms. Phylogenetic analysis revealed that Delta(6)-desaturase genes can be divided into four monophyletic groups. This work paves the way for further study of the functions of Delta(6)-desaturase in fatty acid metabolism and its three-dimensional structure.