Deficiencies in the glucose-6-phosphate transporter (G6PT) cause glycogen storage disease type 1b (GSD-1b), a heritable metabolic disorder. The G6PT protein translocates glucose-6-phosphate from the cytoplasm to the lumen of the endoplasmic reticulum, where glucose-6-phosphatase metabolizes it to glucose and phosphate. Therefore, G6PT and glucose-6-phosphatase work in concert to maintain glucose homeostasis. To delineate the control of G6PT gene expression, we first demonstrated that transcription of the gene requires hepatocyte nuclear factor 1alpha. Consequently, hepatocyte nuclear factor 1alpha-null mice manifest a G6PT deficiency like that of GSD-1b patients. In this study, we delineated the role of glucocorticoids in the transcription of the G6PT gene. We showed that the basal G6PT promoter is contained within nucleotides -369 to -1 upstream of the translation start site, which contains three activation elements. Further, we demonstrated that glucocorticoids activate G6PT transcription and that glucocorticoid action is mediated through a glucocorticoid response element within activation element-2 of the promoter. Taken together, the results suggest that glucocorticoids play a pivotal role in regulating the G6PT gene.