Glutamine (Gln) supplementation in total parenteral nutrition (TPN) has been shown to have a preventive effect on high glucose induced hepatic steatosis. This animal study was undertaken to evaluate whether Gln could prevent hepatic steatosis induced by high fat or high glucose infusion. After placement of internal jugular catheters, rats were divided into three groups: control (n = 8), high fat (n = 13) and high glucose (n = 14) groups. The control group was fed with a chow diet and infused with saline alone. The experimental groups were infused with either a high fat (65% of nonprotein calories) or high glucose (83% of total kilocalories) solution. Energy intake was 35 kcal/100 g body weight per day. TPN solutions were isocaloric, isonitrogenous and isovolemic. Each experimental group was divided into two subgroups, with one receiving a Gln supplement to replace 40% of total amino acid nitrogen. The results demonstrated obvious fatty infiltration in the experimental groups, mainly from triglyceride (TG) accumulation. Plasma very low density lipoprotein-triglyceride (VLDL-TG) was significantly lower in the experimental groups than in the control group, suggesting that liver secretion of TG may have been inhibited in the experimental groups. Liver fatty acid synthetase (FAS) activity and plasma free fatty acid were lower in the high fat group than in the control and high glucose groups. There was no difference in hepatic lipids content, FAS activity, VLDL-TG, hepatic uptake of fatty acids and liver histologic change in the subgroups with and without Gln supplementation. Thus, Gln supplementation in a TPN solution has no effect in preventing hepatic steatosis induced by either high glucose or high fat infusion in rats under these experimental conditions.