Omega-3 fatty acids are essential nutrients and must be supplied in the diets of humans. Fish oil is the major source of omega-3 fatty acids. However, concerns over the quality and sustainability of the fish oil supply have generated interest in supplying omega-3 fatty acids from alternative sources. We report the development of a clean and sustainable source of omega-3 fatty acids by fermentation, using a metabolically engineered strain of the oleaginous yeast Yarrowia lipolytica. Specifically, we will discuss the production of the C20:5n-3 fatty acid eicosapentaenoic acid (EPA). Although some strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, they primarily synthesize linoleic acid (C18:2n-6). We demonstrate that the coordinated expression of desaturase genes and elongase genes comprising omega-3 fatty acid biosynthesis pathways were sufficient for the synthesis of EPA. Using an integrated strategy that involves using strong promoters, multiple copies of exogenous genes, direction of carbon into the engineered pathway, enhancement of acyl-exchange and optimization of fermentation process conditions, we have generated high EPA-producing strains for use in commercial production. This Yarrowia-produced triacylglyceride oil has a unique fatty acid profile, with greater than 55% as EPA and less than 10% as saturated fatty acids. The metabolic engineering of Yarrowia provides a platform technology for producing omega-3 or omega-6 fatty acids of tailored compositions. Thus, our terrestrial production of EPA, DHA, ARA or GLA provides a superior source for these essential molecules for applications in nutritional supplements, functional foods, pharmaceuticals, and animal feeds.