Heparan sulfate (HS), a linear polysaccharide, is involved in diverse biological functions of various tissues. HS is expressed in pancreatic beta-cells and may be involved in beta-cell functions. However, the importance of HS for beta-cell function remains unknown. Here, we generated mice with beta-cell-specific deletion of Ext1 (beta Ext1CKO), which encodes an enzyme essential for HS synthesis, to investigate the detailed roles of HS in beta-cell function. beta Ext1CKO mice decreased body weights compared with control mice, despite increased food intake. Additionally, beta Ext1CKO mice showed impaired glucose tolerance associated with decreased insulin secretion upon glucose challenge. Glucose-induced insulin secretion (GIIS) from isolated beta Ext1CKO islets was also significantly reduced, highlighting the contribution of HS to insulin secretion and glucose homeostasis. The gene expression essential for GIIS was decreased in beta Ext1CKO islets. Pdx1 and MafA were downregulated in beta Ext1CKO islets, indicating that HS promoted beta-cell development and maturation. BrdU- or Ki67-positive beta-cells were reduced in beta Ext1CKO pancreatic sections, suggesting the involvement of HS in the proliferation of beta-cells. Moreover, insufficient vascularization in beta Ext1CKO islets may contribute to central distribution of a-cells. These data demonstrate HS plays diverse roles in beta-cells, and that loss of HS leads to insufficient insulin secretion and dysregulation of glucose homeostasis. (C) 2018 Elsevier Inc. All rights reserved.