Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary alpha-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Delta 6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Delta 6 desaturase activity with both the 18 carbon (C-18) and C-24 n-3 fatty acids, ALA and 24:5 n-3 as well as the C-18 n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Delta 8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Delta 6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.