Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1-/- mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c-/- mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis.