cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N‐terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Δ5‐desaturase of Mortierella alpina. We have confirmed its function as Δ5‐desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Δ5‐desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.