The intestinal brush border fructose transporter GLUT5 (SLC2A5) typically appears in rats after weaning is completed. However, precocious consumption of dietary fructose or in vivo perfusion for 4 h of the small intestine with high fructose (HF) specifically stimulates de novo synthesis of GLUT5 mRNA and protein before weaning is completed. Intermediary signals linking the substrate, fructose, to GLUT5 transcription are not known but should also respond to fructose perfusion. Hence, we used microarray hybridization and RT-PCR to identify genes whose expression levels change during HF relative to high-glucose (HG) perfusion. Expression of GLUT5 and NaPi2b, the intestinal Na⁺-dependent phosphate transporter, dramatically increased and decreased, respectively, with HF perfusion for 4 h. Expression of >20 genes, including two key gluconeogenic enzymes, glucose-6-phosphatase (G6P) and fructose-1,6-bisphosphatase, also increased markedly, along with fructose-2,6-bisphosphatase, an enzyme unique to fructose metabolism and regulating fructose-1,6-bisphosphatase activity. GLUT5 and G6P mRNA abundance, which increased dramatically with HF relative to HG, α-methylglucose, and normal Ringer perfusion, may be tightly and specifically linked to changes in intestinal luminal fructose but not glucose concentrations. G6P but not GLUT5 mRNA abundance increased after just 20 min of HF perfusion. This cluster of gluconeogenic enzymes and their common metabolic intermediate fructose-6-phosphate may regulate fructose metabolism and GLUT5 expression in the small intestine.