Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.