Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.