Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are nutritionally important long-chain (≥ C20) omega-3 polyunsaturated fatty acids (ω3 LC-PUFA) currently obtained mainly from marine sources. A set of genes encoding the fatty acid chain elongation and desaturation enzymes required for the synthesis of LC-PUFA from their C18 PUFA precursors was expressed seed-specifically in Arabidopsis thaliana. This resulted in the synthesis of DHA, the most nutritionally important ω3 LC-PUFA, for the first time in seed oils, along with its precursor EPA and the ω6 LC-PUFA arachidonic acid (ARA). The assembled pathway utilised Δ5 and Δ6 desaturases that operate on acyl-CoA substrates and led to higher levels of synthesis of LC-PUFA than previously reported with acyl-PC desaturases. This demonstrates the potential for development of land plants as alternative sources of DHA and other LC-PUFA to meet the growing demand for these nutrients.