Long Chain Polyunsaturated Fatty Acids (PUFAs), such as eicosapentaenoic and docosahexaenoic acid, have gained recognition as being important in the treatment and prevention of a variety of human disorders and diseases, and as such have huge potential in both the nutritional supplement and function food markets. Consequently, suitable sources of these compounds have been of interests to scientists for many years. Isolation of highly oleaginous microorganisms, such as Thraustochytrium sp. ONC-T18, has led to the development of fermentation technologies as the favored method due to its purity and commercial sustainability. Nevertheless, efficient microbial PUFAs production targeting specific fatty acids requires knowledge of the mechanisms by which biosynthesis is accomplished within the cell. Although the main aspect of PUFA accumulation and the two microbial PUFA biosynthesis pathways (fatty acid and polyketide-like PUFA synthases) have been known, the metabolic specificities of these pathways are still unclear. In order to elucidate fatty acid biosynthesis complexes from various sources within Thraustochytrium sp. ONC-T18, compounds that regulate PUFA metabolism such as nutrients, enzyme inhibitors, as well as chemical mutagens were investigated. Together with genetic and biochemical information, the application of these regulating compounds provides an insight into the mechanism by which specific fatty acids produced.