Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate-amidotransferase (GFAT) as rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine transforming growth factor β1 (TGF-β1) in fibrotic complications, particularly in diabetic kidney disease. Here, we investigate in a stable transfection system the effect of overexpression of GFAT on TGF-β1 synthesis in NIH-3T3 fibroblasts. We demonstrate a 1.8-fold stimulation of TGF-β1 mRNA and a 1.9-fold increased protein expression, whereas TGF-β2 production was not upregulated. The 1.5-fold enhanced TGF-β1 promoter activity suggests a transcriptional regulation. The elevated TGF-β1 protein is biologically active since GFAT-overexpressing cells exhibit a 2-fold fibronectin production. The results indicate a GFAT-dependent induction of TGF-β1 synthesis.