Abstract Background Therapeutic replacement of pancreatic endocrine β-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to β-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase. Results Human pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as β-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response. Conclusion The results of this study serve as a proof of concept for a probable source of β-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.