Marine ecosystems are responsible for virtually all production of omega-3 (ω3) long-chain polyunsaturated fatty acids (PUFA), which are essential nutrients for vertebrates. Current consensus is that marine microbes account for this production, given their possession of key enzymes including methyl-end (or “ωx”) desaturases. ωx desaturases have also been described in a small number of invertebrate animals, but their precise distribution has not been systematically explored. This study identifies 121 ωx desaturase sequences from 80 species within the Cnidaria, Rotifera, Mollusca, Annelida, and Arthropoda. Horizontal gene transfer has contributed to this hitherto unknown widespread distribution. Functional characterization of animal ωx desaturases provides evidence that multiple invertebrates have the ability to produce ω3 PUFA de novo and further biosynthesize ω3 long-chain PUFA. This finding represents a fundamental revision in our understanding of ω3 long-chain PUFA production in global food webs, by revealing that numerous widespread and abundant invertebrates have the endogenous capacity to make significant contributions beyond that coming from marine microbes.