The transplantation of insulin-producing cells is a promising approach for the treatment of insulin-dependent diabetes mellitus; however, lack of pancreas donors limits its application. Pancreatic duodenal homeobox 1 (Pdx1) plays a key role in the differentiation of various non-β-cells into insulin-producing cells, but the potential mechanism remains to be clarified. The purpose of this study was to confirm that the expression of Pdx1 could mediate the differentiation of rat mesenchymal stem cells (MSCs) into insulin-producing cells and evaluate the potential molecular mechanisms in the process that Pdx1 activates transcription of insulin gene. In this study, glucose-stimulated insulin secretion was obviously detected in MSCs transfected with Pdx1 cDNA by insulin release assay and the islet-like structure formed in Pdx1-expressing MSCs was stained into black-red by dithizone, while the native MSCs were opposite. In addition, we uncovered the close relationships among the expression of Pdx1, insulin and Ngn3 genes, whose expression indicated parallel changes after high glucose challenge, and the fluctuation of Pdx1 and Ngn3 partly resulted in the unstable release of insulin. Taken together, these findings demonstrated that the effective role of Pdx1 gene in inducing insulin-producing cells, which may shuttle to the nucleoplasm of MSCs under high glucose, then initiate the expression of native transcription factors Ngn3 and recruit other proteins, resulting in transactivation of the relevant genes including insulin and generation of β cell phenotype. Accordingly, these results would provide new insights that may be applicable to improve β cell replacement strategies and enhance diabetes therapy in the future.