NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic β cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties. Overall design: Mouse pancreatic islets were isolated (and pooled) from four mice per each genotype (control and Neurod1CKO) by perfusion of the common bile duct with Collagenase. Digestion of islets to single cell was performed with trypsin supplemented with Actinomycin D. Single-cell suspension was used for library preparation:743 Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10 × Genomics, Pleasanton, CA) was sed to prepare the sequencing libraries, according to the manufacturer’s protocols.