Context: Dysregulated chylomicron metabolism may account for hypertriglyceridemia and increased risk of cardiovascular disease in obese subjects. Omega-3 fatty acid ethyl ester (ω-3 FAEE) supplementation decreases plasma triglyceride. However, its effect on postprandial chylomicron metabolism in obese subjects on a weight loss diet has not yet been investigated. Objective: We aimed to examine the effect of ω-3 FAEE supplementation on apolipoprotein (apo) B-48 kinetics in obese subjects on a weight loss diet. Design, Setting and Patients: We carried out a 12-week, randomized trial of a hypocaloric diet plus 4g/day ω-3 FAEE supplementation (46% eicosapentaenoic acid and 38% docosahexaenoic acid) (n=13) compared with a hypocaloric diet alone (n=12) on postprandial apoB-48 kinetics in obese subjects following ingestion of an oral load. ApoB-48 kinetics were determined using stable isotope tracer kinetics and multi-compartmental modeling. Outcomes Measures: Plasma total and incremental apoB-48 0-10hr area-under-the-curves (AUC), as well as apoB-48 secretion and fractional catabolic rate. Results: Weight loss with or without ω-3 FAEE supplementation significantly reduced body weight, total fat mass, HOMA score, fasting triglyceride concentration, postprandial triglyceride AUC, and increased plasma HDL-cholesterol concentration (P<0.05 in all). Compared with weight loss alone, weight loss plus ω-3 FAEE significantly (all P<0.05) decreased fasting triglyceride (-11%), apoB-48 (-36%) concentrations, postprandial triglyceride (-21%) and apoB-48 (-22%) total AUCs, as well as incremental postprandial triglyceride AUCs (-32%). ω-3 FAEE also significantly decreased apoB-48 secretion at basal state, without significant effect during the postprandial period (3-6hr). The fractional catabolic rate of apoB-48 increased, with both interventions with no significant independent effect of ω-3 FAEE supplementation. Conclusion: Addition of n-3 FAEE supplementation to a moderate weight loss diet in obese subjects can significantly improve chylomicron metabolism by independently decreasing the secretion of apoB-48.