The pancreatic and duodenal homeobox gene 1 (Pdx-1) plays a key role in normal pancreas development and is required for maintaining the normal function of islets. In this study, we examined whether human adipose tissue-derived stem cells (hASCs) could differentiate into insulin-producing cells by exogenously expressed Pdx-1. hASCs were infected with recombinant adenovirus encoding the mouse Pdx-1 gene and differentiated under high-glucose conditions. Insulin transcript levels and the expression of key transcription factors required for pancreatic development including FoxA2, Nkx2.2, and NeuroD were significantly increased by exogenous Pdx-1 overexpression. The expression of Nkx6.1 was found only in Pdx-1-induced hASCs. In addition to transcripts for transcription factors involved in pancreatic development, transcripts for the GLP-1 receptor, glucokinase, and glucose transporter, which are required for maintaining the function of pancreatic β-cells, were observed only in Pdx-1-induced hASCs. Pdx-1-induced hASCs exhibited insulin secretion in response to glucose challenge in vitro. When Pdx-1-induced hASCs were transplanted into streptozotocin (STZ)-induced diabetic mice, they reduced blood glucose levels, although they did not restore normoglycemia. These results demonstrate that the expression of exogenous Pdx-1 is sufficient to induce pancreatic differentiation in vitro but does not induce the fully functional, mature insulin-producing cells that are required for restoring normoglycemia in vivo.