Polyunsaturated fatty acid (PUFA) supplements have been trialled as a treatment for a number of conditions and produced a variety of results. This variety is ascribed to the supplements, that often comprise a mixture of fatty acids, and to different effects in different organs. In this study, we tested the hypothesis that the supplementation of individual PUFAs has system-level effects that are dependent on the molecular structure of the PUFA. We undertook a network analysis using Lipid Traffic Analysis to identify both local and system-level changes in lipid metabolism using publicly available lipidomics data from a mouse model of supplementation with FA(20:4n-6), FA(20:5n-3), and FA(22:6n-3); arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively. Lipid Traffic Analysis is a new computational/bioinformatics tool that uses the spatial distribution of lipids to pinpoint changes or differences in control of metabolism, thereby suggesting mechanistic reasons for differences in observed lipid metabolism. There was strong evidence for changes to lipid metabolism driven by and dependent on the structure of the supplemented PUFA. Phosphatidylcholine and triglycerides showed a change in the variety more than the total number of variables, whereas phosphatidylethanolamine and phosphatidylinositol showed considerable change in both which variables and the number of them, in a highly PUFA-dependent manner. There was also evidence for changes to the endogenous biosynthesis of fatty acids and to both the elongation and desaturation of fatty acids. These results show that the full biological impact of PUFA supplementation is far wider than any single-organ effect and implies that supplementation and dosing with PUFAs require a system-level assessment.