Diabetes investigators have been interested in the regulation of glucokinase for years because this enzyme catalyzes the initial step in glucose metabolism by the liver and pancreatic β-cell. However, now that mutations in the glucokinase gene have been associated with early-onset noninsulin-dependent diabetes mellitus, there is increased interest in the mechanisms that enable the gene to be differentially regulated in a cell-specific manner. In this chapter, we provide an overview of our current knowledge of the mechanisms by which the glucokinase gene is regulated and how glucokinase gene mutations might cause the hyperglycemia associated with certain types of diabetes.