Direct reprogramming of autologous cells from diabetes patients to insulin producing cells is a new method for pancreatic cell replacement therapy. At present, transdifferentiation among mature cells is achieved mainly by introducing foreign genes into the starting tissue with viral vector, but there are potentical safety problems. In the present study, we delivered plasmids carrying Pdx1, Neurog3 and MafA genes (PNM) into mouse hepatocytes by hydrodynamics tail vein injection, investigated islet β cells markers in transfected cells from protein and mRNA level, and then observed the long-term control of blood glucose in diabetic mice. We found that hepatocytes could be directly reprogrammed into insulin-producing cells after PNM gene transfection by non-viral hydrodynamics injection, and fasting blood glucose was reduced to normal, and lasted until 100 days after transfection. Intraperitoneal glucose tolerance test (IPGTT) showed that glucose regulation ability was improved gradually and the serum insulin level approached to the level of normal mice with time. Insulin-positive cells were found in the liver tissue, and the expression of various islet β-cell-specific genes were detected at the mRNA level, including islet mature marker gene Ucn3. In conclusion, we provide a new approach for the treatment of diabetes by in vivo direct reprogramming of liver cells to insulin producing cells through non-viral methods.