--- - |2- Abstract Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell‐based treatment approaches. However, severe shortage of donor islets–pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell–based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton’s jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine‐like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum‐free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor‐like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin‐producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX‐1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269‐treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin‐producing cells. - Journal of Cellular Physiology, EarlyView.