Eicosapentaenoic acid (EPA) is an ω3 polyunsaturated fatty acid which has been demonstrated to play important roles in a number of aspects of human health. EPA is traditionally obtained from marine fish oils. However, the shrinking fish populations are making the sustainability of these sources questionable. Consequently, alternative sources of EPA are being sought, especially from marine microalgae, bacteria, and fungi. These microorganisms contain relatively large amounts of high-quality EPA and they are the primary producers of this important fatty acid. There are two distinct pathways for EPA de novo biosynthesis in microbial systems: the desaturation and elongation pathway and the polyketide pathway. Genes involved in the biosynthetic pathways have been identified from different microorganisms and characterized in depth. In addition, numerous strategies have been developed for commercial production of EPA by microbial fermentation, among which strain improvements by genetic engineering could provide high-yield producers of EPA. In this review, we summarize recent efforts and experiences devoted to metabolic engineering of various microorganisms that lead to efficient biocatalysts for the production of EPA, as well as the key limitations and challenges. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of EPA biosynthesis and a greater potential of microbial production. Continued advances in metabolic engineering will help to improve the final titer, productivity, and yield of EPA.