Glucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia.