It has been indicated that the combination of pancreatic and duodenal homeobox 1 (Pdx1), MAF bZIP transcription factor A (MafA) and neurogenin 3 (Ngn3) was able to reprogram various cell types towards pancreatic beta-like cells (p beta LCs). Paired box 4 (Pax4), a transcription factor, has a key role in regulating the maturation of pancreatic beta-cells (p beta Cs). In the present study, it was investigated whether Pax4 is able to synergistically act with Pdx1, Ngn3 and MafA to induce human umbilical cord mesenchymal stem cells (HuMSCs) to differentiate into functional p beta Cs in vitro. HuMSCs were isolated, cultured and separately transfected with adenovirus (Ad) expressing enhanced green fluorescence protein, Pax4 (Ad-Pax4), Pdx1+MafA+Ngn3 (Ad-3F) or Ad-Pxa4 + Ad-3F. The expression of C-peptide, insulin and glucagon was detected by immunofluorescence. The transcription of a panel of genes was determined by reverse transcription-quantitative PCR, including glucagon (GCG), insulin (INS), NK6 homeobox 1 (NKX6-1), solute carrier family 2 member 2 (SLC2A2), glucokinase (GCK), proprotein convertase subtilisin/kexin type 1 (PCSK1), neuronal differentiation 1 (NEUROD1), ISL LIM homeobox 1 (ISL 1), Pax6 and PCSK type 2 (PCSK2). Insulin secretion stimulated by glucose was determined using ELISA. The results suggested that, compared with Ad-3F alone, cells co-transfected with Ad-Pax4 and Ad-3F expressed higher levels of INS and C-peptide, as well as genes expressed in pancreatic beta precursor cells, and secreted more insulin in response to high glucose. Furthermore, the expression of GCG in cells transfected with Ad-3F was depressed by Ad-Pax4. The present study demonstrated that Pax4 was able to synergistically act with the transcription factors Pdx1, Ngn3 and MafA to convert HuMSCs to functional p beta LCs. HuMSCs may be potential seed cells for generating functional p beta LCs in the therapy of diabetes.