Background: Recent studies suggest that eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have distinct effects on cardiometabolic risk factors. The Omega-3 Index (O3I), which is calculated as the proportion of EPA and DHA in red blood cell (RBC) membranes, has been inversely associated with the risk of coronary heart diseases and coronary mortality. The objective of this study was to compare the effects of EPA and DHA supplementation on the O3I in men and women with abdominal obesity and subclinical inflammation. Methods: In a double-blind controlled crossover study, 48 men and 106 women with abdominal obesity and subclinical inflammation were randomized to a sequence of three treatment phases: 1–2.7 g/d of EPA, 2–2.7 g/d of DHA, and 3–3 g/d of corn oil (0 g of EPA+DHA). All supplements were provided as 3×1 g capsules for a total of 3 g/d. The 10-week treatment phases were separated by nine-week washouts. RBC membrane fatty acid composition and O3I were assessed at baseline and the end of each phase. Differences in O3I between treatments were assessed using mixed models for repeated measures. Results: The increase in the O3I after supplementation with DHA (+5.6% compared with control, P < 0.0001) was significantly greater than after EPA (+3.3% compared with control, P < 0.0001; DHA vs. EPA, P < 0.0001). Compared to control, DHA supplementation decreased (−0.8%, P < 0.0001) while EPA increased (+2.5%, P < 0.0001) proportion of docosapentaenoic acid (DPA) in RBCs (DHA vs. EPA, P < 0.0001). The baseline O3I was higher in women than in men (6.3% vs. 5.8%, P=0.011). The difference between DHA and EPA in increasing the O3I tended to be higher in men than in women (+2.6% vs. +2.2% respectively, P for the treatment by sex interaction=0.0537). Conclusions: The increase in the O3I is greater with high dose DHA supplementation than with high dose EPA, which is consistent with the greater potency of DHA to modulate cardiometabolic risk factors. The extent to which such differences between EPA and DHA in increasing the O3I relates to long-term cardiovascular risk needs to be investigated in the future.