Heparan sulfate (HS) binds with several signaling molecules and regulates ligand–receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet β-cells after 1week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in β-cells. These mice exhibited abnormal islet morphology with reduced β-cell proliferation after 1week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.