Type 1 and type 2 diabetes result from a deficit in insulin production and β-cell mass. Methods to expand β-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote β-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [3H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates β-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating β-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [3H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human β-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [3H]thymidine incorporation. CCK is a β-cell proliferative factor that is effective in both mouse and human islets. CCK triggers β-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.