Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells. © The Author(s) 2018.