BACKGROUND. Dietary changes have led to a growing prevalence of Type 2 diabetes and non-alcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite high protein diets being popular for weight loss to tackle these metabolic disorders, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterise the effect of dietary protein on de novo lipid synthesis. METHODS. Herein, we use a 3-way crossover interventional study in healthy males to determine the effect of high protein feeding on de novo lipogenesis as well as in vitro models to determine the effects of specific amino acids on fatty acid synthesis. The primary outcome was change in de novo lipogenesis-associated triglycerides in response to protein feeding. RESULTS. We demonstrate that high protein feeding, rich in glutamate, increases de novo lipogenesis-associated triglycerides in plasma (1.5-fold compared to Control; p < 0.0001) and liver-derived very low-density lipoprotein particles (1.8 fold; p < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate derived carbon is incorporated into palmitate and subsequently into triglycerides. In addition, supplementation with glutamate, glutamine and leucine, but not lysine increases synthesised triglyceride content in cells and decreases glucose uptake. Glutamate, glutamine and leucine increase activation of protein kinase B, suggesting that these amino acids induce de novo lipogenesis via the insulin signalling cascade. CONCLUSION. These findings provide mechanistic insight into how select amino acids may induce de novo lipogenesis and insulin resistance, suggesting that high protein feeding to tackle diabetes and obesity requires greater consideration.