Exendin-4 is a stable peptide agonist of GLP-1 receptor that exhibits insulinotropic actions. Some in vivo studies indicated insulin-independent glucoregulatory actions of exendin-4. That finding prompted us to evaluate effects of exendin-4 on liver glucose metabolism. Acute and chronic treatment of exendin-4 resulted in increased hepatic glucokinase activity in db/db mice but not in lean C57 mice. The stimulatory effect of exendin-4 on glucokinase activity was abrogated by exendin 9-39, a GLP-1 antagonist. Exposure of hepatocytes isolated from db/db mice to exendin-4 elicited a rapid increase in cAMP, which was synergized by IBMX, an inhibitor of cAMP degradation. The GLP-1 antagonist, exendin 9-39, has abolished the cAMP generating effects of exendin-4 as well. Furthermore, chronic treatment of exendin-4 in streptozotocin-treated C57 mice resulted in restoration of hepatic glycogen, an indicator of improved glucose metabolism, without apparent changes in serum insulin levels. In conclusion, exendin-4 increased glucokinase enzyme protein and activity in liver via a mechanism parallel to and independent of insulin. Exendin-4-induced increase in hepatic glucokinase activity is more pronounced in the presence of hepatic insulin resistance. This beneficial effect of exendin-4 on liver glucokinase activity may be mediated by GLP-1 receptor.