Objective To evaluate the effects of key insulin gene transcription regulators (PDX-1, NeuroD1 and MafA) on the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells. Methods Mouse embryonic fibroblasts (MEFs) were infected with lentivirus (LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection, and iPSCs were selected and identified. Then the iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad-mNeuroD1-IRES -GFP and Ad-mMafA-IRES -GFP) to induce differentiation into insulin-producing cells in vitro. RT-PCR was applied to detect expression of functional genes in pancreatic islet B cells; immunofluorescence was used to examine the expression and location of insulin protein; and ELISA was used to determine the volumes of secreted insulin at different concentrations of glucose (0,5,10,20,30, and 40 mmol/L). Results The iPSCs derived from MEFs could form intensive clones with smooth boundary, express embryonic stem cell-specific cell surface markers, including Nanog, SSEA-1 and Rex-1, and differentiate into three embryonic layers, which indicating that MEFs were successfully reprogrammed into iPSCs. Mouse iPSCs infected with Ad-PDX-1-IRES-GFP, Ad-mNeuroD-IRES-GFP, and Ad-mMafA-IRES-GFP could differentiate into pancreatic islet B cells. RT-PCR results showed that polygene-modified iPSCs and pancreatic islet B-cell line MIN6 had similar gene expression profile. Immunofluorescence analyses confirmed insulin expression in the differentiated cells. Results of ELISA showed that polygene-modified iPSCs had a satisfactory response to different concentrations of glucose. Conclusion Key insulin gene transcription regulators PDX-1, NeuroD1 and MafA can work synergistically to induce mouse iPSCs differentiation into pancreatic islet B cells capable of insulin biosynthesis and secretion.