We previously identified an E-box to be implicated in high-glucose–induced transforming growth factor-β1 (TGF-β1) gene stimulation in murine mesangial cells. In the present study, we evaluated the role of upstream stimulatory factors (USFs) in mediating glucose-induced stimulation of TGF-β1. Mesangial cells cultured in glucose concentrations exceeding 2.7 mmol/l d-glucose exhibited increased levels of USF1 and USF2 protein by Western analysis and electrophoretic mobility shift assay (EMSA). An E-box element from the murine TGF-β1 promoter revealed USF1 and USF2 binding by EMSA. Chromatin immunoprecipitation assay revealed in vivo binding of USF1 to a glucose-responsive region of the TGF-β1 promoter. Transient cotransfection studies of 293 cells with USF1 led to a twofold increase in TGF-β1 promoter activity and a 46% increase in secreted TGF-β1 protein levels. Wild-type and USF2 knockout mice exhibited a 2.5-fold stimulation of renal TGF-β1 expression upon fasting and refeeding with a carbohydrate-rich diet, whereas USF1 knockout mice exhibited only a minimal increase of renal TGF-β1 upon refeeding. USF1 mRNA levels were increased in mouse kidneys with carbohydrate refeeding, and USF1 protein was increased in diabetic rat kidneys compared with controls. We conclude that USF1 is stimulated by modest increases in glucose concentration in murine mesangial cells, bind to the murine TGF-β1 promoter, contribute to carbohydrate-induced renal TGF-β1 expression, and may play a role in diabetes-related gene regulation in the kidney.