The Delta(6)-fatty acid desaturase is a key enzyme in the synthesis of an important fatty acid, gamma-linolenic acid. We have characterized, by heterologous expression in Saccharomyces cerevisiae, substrate specificity and preference of Delta(6)-desaturase of Mucor rouxii. Fatty acid supplementation was carried out based on the predicted enzyme topology, fatty acid phenotype and the corresponding metabolic pathway in M. rouxii. The enzyme has a broad substrate specificity as based on C15-C18. The result also supported classification of the M. rouxii Delta(6)-desaturase into a front-end desaturase. Interestingly, a relatively rare activity based on odd acyl chains and not described previously in other eukaryotic Delta(6)-desaturases was also observed.