Surrogate β-cells derived from available adult stem cells are urgently needed for the treatment of insulin-deficient diabetes, and hepatocytes constitute an attractive alternative. Herein, we attempted to generate insulin-pro-ducing cells from adult mouse primary hepatocytes (HCs) using triple adenoviruses harboring PDX-1/VP-16, BETA2, and MafA. We noted characteristic changes in the transduced HCs into pancreatic β-cells, including reduced albumin gene and increased insulin, insulin content, and the expression of a variety of pancreatic genes. Glu-cose tolerance and survival are improved by the renal capsular transplantation. These data demonstrated that the transdifferentiation of HCs into insulin-producing cells could be achieved under both in vitro and in vivo conditions. Further, these data suggest that induction of insulin-producing cells from liver provides a potential cell-replacement therapy for the treatment of patients with diabetes using alternative transplantable cell source.