Dysregulated VLDL-TAG (very-low-density lipoprotein triacylglycerol) metabolism in obesity may account for hypertriacylglycerolaemia and increased cardiovascular disease. ω-3 FAEEs (omega-3 fatty acid ethyl esters) decrease plasma TAG and VLDL concentrations, but the mechanisms are not fully understood. In the present study, we carried out a 6-week randomized, placebo-controlled study to examine the effect of high-dose ω-3 FAEE supplementation (3.2 g/day) on the metabolism of VLDL-TAG in obese men using intravenous administration of d5-glycerol. We also explored the relationship of VLDL-TAG kinetics with the metabolism of VLDL-apo (apolipoprotein) B-100 and HDL (high-density lipoprotein)-apoA-I. VLDL-TAG isotopic enrichment was measured using gas chromatography-mass spectrometry. Kinetic parameters were derived using a multicompartmental model. Compared with placebo, ω-3 FAEE supplementation significantly lowered plasma concentrations of total (-14%, P<0.05) and VLDL-TAG (-32%, P<0.05), as well as hepatic secretion of VLDL-TAG (-32%, P<0.03). The FCR (fractional catabolic rate) of VLDL-TAG was not altered by ω-3 FAEEs. There was a significant association between the change in secretion rates of VLDL-TAG and VLDL-apoB-100 (r=0.706, P<0.05). However, the change in VLDL-TAG secretion rate was not associated with change in HDL-apoA-I FCR (r=0.139, P>0.05). Our results suggest that the TAG-lowering effect of ω-3 FAEEs is associated with the decreased VLDL-TAG secretion rate and hence lower plasma VLDL-TAG concentration in obesity. The changes in VLDL-TAG and apoB-100 kinetics are closely coupled.