A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty rats (ZDF). We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF. We measured expression and localization of GK and GK-regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic-hyperinsulinemic clamp after 1 or 6 weeks of treatment with sodium-glucose transporter 2 inhibitor used to correct the persistent hyperglycemia of ZDF. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF was associated with the co-residency of GKRP with GK in the cytoplasm in the mid stage of diabetes, which was followed by a decrease on GK protein levels due to impaired post-transcriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal condition and normalizing translocation of GK from the nucleus to the cytoplasm with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver's metabolic inability to respond to hyperglycemic-hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness on hepatic glucose flux by affecting GK regulation in the ZDF.