Arbuscular mycorrhizal (AM) fungi are oleaginous organisms, and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1Δ11cis ). However, it is not known how this uncommon fatty acid species is made. Here, we have cloned two homologues of lepidopteran fatty acyl-coenzyme A Δ11 desaturases from the AM fungus Rhizophagus irregularis. Both enzymes, DES1 and DES2, are expressed in intraradical mycelium and can complement the unsaturated fatty acid-requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1Δ9cis ) production, whereas DES2 expression results in the production of 16:1Δ11cis and vaccenic acid (18:1Δ11cis ). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1Δ11cis in R. irregularis.,© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.