We have previously demonstrated that dietary saturated fatty acids (SFA), when compared to polyunsaturated fatty acids (PUFA), are preferentially partitioned into oxidation pathways. However, it remains unclear if this preferential handling is maintained when hepatocellular metabolism is shifted toward fatty acid (FA) esterification and away from oxidation, such as when hepatic de novo lipogenesis (DNL) is upregulated. To investigate whether an acute upregulation of hepatic DNL influences dietary FA partitioning into oxidation pathways. 20 healthy volunteers (11 females) underwent a fasting baseline visit followed by two study days, 2-weeks apart. Prior to each study day, participants consumed an isocaloric high-carbohydrate diet (to upregulate hepatic DNL) for 3-days. On the two study days, participants consumed an identical standardised test meal that contained either [U13C]palmitate or [U13C]linoleate, in random order, to trace the fate of dietary FA. Blood and breath samples were collected over a 6h postprandial period and 13C enrichment in breath CO2 and plasma lipid fractions were measured using gas-chromatography-combustion-isotope ratio mass spectrometry. Compared to the baseline visit, fasting plasma triglyceride concentrations and markers of hepatic DNL, the lipogenic and stearyl-CoA desaturase indices, were significantly (p<0.05) increased after consumption of the high-carbohydrate diet. Appearance of 13C in expired CO2 and tracer recovery were significantly (p<0.05) higher after consumption of the meal containing [U13C]linoleate compared to [U13C]palmitate (5.1±0.5% vs. 3.7±0.4%), respectively. Incorporation of 13C into the plasma triglyceride and non-esterified fatty acid pool was significantly (p<0.001) greater for [U13C]palmitate compared to [U13C]linoleate. Dietary PUFA compared to SFA appear to be preferentially partitioned into oxidation pathways during an acute upregulation of hepatic DNL, thus consumption of a PUFA-enriched diet may help mitigate intrahepatic triglyceride accumulation in individuals at risk of cardiometabolic disease.