A high fructose intake exacerbates postprandial plasma triacylglycerol (TAG) concentration, an independent risk factor for cardiovascular disease, although it is unclear whether this is due to increased production or impaired clearance of triacylglycerol (TAG)-rich lipoproteins. We determined the in vivo acute effect of fructose on postprandial intestinal and hepatic lipoprotein TAG kinetics and de novo lipogenesis (DNL). Five overweight men were studied twice, 4 weeks apart. They consumed hourly mixed-nutrient drinks that were high-fructose (30% energy) or low-fructose (<2% energy) for 11 h. Oral 2H2O was administered to measure fasting and postprandial DNL. Postprandial chylomicron (CM)-TAG and very low-density lipoprotein (VLDL)-TAG kinetics were measured with an intravenous bolus of [2H5]-glycerol. CM and VLDL were separated by their apolipoprotein B content using antibodies. Plasma TAG (p < 0.005) and VLDL-TAG (p = 0.003) were greater, and CM-TAG production rate (PR, p = 0.046) and CM-TAG fractional catabolic rate (FCR, p = 0.073) lower when high-fructose was consumed, with no differences in VLDL-TAG kinetics. Insulin was lower (p = 0.005) and apoB48 (p = 0.039), apoB100 (p = 0.013) and non-esterified fatty acids (NEFA) (p = 0.013) were higher after high-fructose. Postprandial hepatic fractional DNL was higher than intestinal fractional DNL with high-fructose (p = 0.043) and low-fructose (p = 0.043). Fructose consumption had no effect on the rate of intestinal or hepatic DNL. We provide the first measurement of the rate of intestinal DNL in humans. Lower CM-TAG PR and CM-TAG FCR with high-fructose consumption suggests lower clearance of CM, rather than elevated production, may contribute to elevated plasma TAG, possibly due to lower insulin-mediated stimulation of lipoprotein lipase.