Background: While beneficial health effects of fish and fish oil consumption are well documented, the incorporation of n-3 polyunsaturated fatty acids in plasma lipid classes is not completely understood. The aim of this study was to investigate the effect of fish oil supplementation on the plasma lipidomic profile in healthy subjects. Methodology/Principal Findings: In a double-blinded randomized controlled parallel-group study, healthy subjects received capsules containing either 8 g/d of fish oil (FO) (1.6 g/d EPA+DHA) (n = 16) or 8 g/d of high oleic sunflower oil (HOSO) (n = 17) for seven weeks. During the first three weeks of intervention, the subjects completed a fully controlled diet period. BMI and total serum triglycerides, total-, LDL- and HDL-cholesterol were unchanged during the intervention period. Lipidomic analyses were performed using Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (QTOFMS), where 568 lipids were detected and 260 identified. Both t-tests and Multi-Block Partial Least Square Regression (MBPLSR) analysis were performed for analysing differences between the intervention groups. The intervention groups were well separated by the lipidomic data after three weeks of intervention. Several lipid classes such as phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylglycerol, and triglycerides contributed strongly to this separation. Twenty-three lipids were significantly decreased (FDR<0.05) in the FO group after three weeks compared with the HOSO group, whereas fifty-one were increased including selected phospholipids and triglycerides of long-chain polyunsaturated fatty acids. After seven weeks of intervention the two intervention groups showed similar grouping. Conclusions/Significance: In healthy subjects, fish oil supplementation alters lipid metabolism and increases the proportion of phospholipids and triglycerides containing long-chain polyunsaturated fatty acids. Whether the beneficial effects of fish oil supplementation may be explained by a remodeling of the plasma lipids into phospholipids and triglycerides of long-chain polyunsaturated fatty acids needs to be further investigated.