High intakes of fructose are associated with hypertriglyceridemia attributed primarily to hepatic conversion of fructose to fats. Even though the small intestine expresses lipogenic genes, is luminally exposed to high fructose, and can convert labelled fructose to fatty acids, there is little information on the effect of fructose on expression of small intestinal lipogenic genes, and on the identity of intestinal cell type(s) capable of fructose‐induced regulation. We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and chylomicron synthesis and that regulation depends on the fructose transporter GLUT5 and on the enzyme ketohexokinase (KHK). Because intestinal stem cells can differentiate into absorptive enterocytes and secretory goblet, Paneth and other cells, we next hypothesized that fructose regulation of lipogenic genes is specific to enterocytes and not to other cell types. We fed for 3 d fructose or glucose solutions by gavage to wildtype, GLUT5‐KO and KHK‐KO mice with free access to food, then expression of representative genes involved in lipid and chylomicron synthesis was determined. We then isolated stem cells from chow‐fed wildtype, GLUT5‐KO and KHK‐KO mice and directed the differentiation of these cells into organoids highly enriched in enterocytes (90%), goblet (80%), Paneth (65%) and stem (90%) cells, incubated them in 10 mM fructose or glucose solutions, then determined mRNA levels of lipogenic genes. In vivo mRNA and protein expression of representative genes involved in lipid synthesis (sterol responsive element binding protein‐1c, acetyl‐CoA carboxylase 1, fatty acid synthase, stearoyl‐CoA desaturase 1, diacylglycerol acyltransferase 1 and glycerol kinase) and intestine‐specific apolipoproteins APO‐A4 and APO‐B48 each increased markedly in wildtype, but not in GLUT5‐KO and KHK‐KO, mice fed fructose. In contrast, expression of the nonfructolytic nonlipogenic glucose transporter SGLT1 was independent of diet. GLUT5‐ and KHK‐dependent, fructose‐induced increases in mRNA levels of these genes were observed only in enterocytes in vitro. Dietary fructose specifically stimulates the expression of lipogenic and apolipoprotein genes in the small intestine, and these effects require fructose transport and metabolism. Thus, ingested fructose must be absorbed by GLUT5 and not merely “tasted” by numerous sweet receptors expressed in the gut, then phosphorylated by KHK to fructose‐1‐phosphate. Moreover, fructose effects occur only in absorptive enterocytes, suggesting that the capacity for fructose modulation of these genes is lost during differentiation into secretory cell types.Support or Funding InformationSupported by NSF Grant IOS‐1456673 (RPF) and by NIH Grant R01‐DK‐102934 (NG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.