The pancreas and the liver share the same endodermal origin. We have been studying whether mature hepatocytes can be induced to differentiate into pancreatic beta-cells by in vitro delivery of transcriptional factors using a non-viral approach. Here we showed that nucleofection allowed suitable transfection of primary hepatocytes employing various non-viral methods. We introduced either pancreatic and duodenal homeobox 1 (Pdx1) or neurogenin 3 (Ngn3), or both, into the mature cells using nucleofection. Co-expression of pdx1 and ngn3 using a bicistronic vector activated the transcription of various islet-related genes, and the transfected hepatocytes acquired the ability to synthesize and secrete insulin. Our results suggest that simultaneous expression of Pdx1 and Ngn3 is an excellent inducer of liver-to-pancreas reprogramming, and that reprogramming will occur even in mature somatic cells without the need for viral vectors. These findings are of considerable significance for further therapeutic development for various intractable diseases including diabetes.