Septin5 interacts with SNARE proteins to regulate exocytosis in neurons, but its role in pancreatic β-cells is unknown. Here, we report that Septin5 is abundant in rodent and human β-cells, deletion of which dramatically enhances biphasic glucose-stimulated insulin secretion, including in type 2 diabetes (T2D). Super-resolution imaging shows that Septin5 is preferentially assembled in microtubule-plasma membrane contact sites in a microtubule-dependent manner, which provides discrete harbor for secretory granule anchoring. By decreasing the stability of the cortical microtubule meshwork, Septin5 depletion increases insulin granule dynamics and access to the plasma membrane. Analysis of spatiotemporal coupling of fusion events and localized Ca(2+) influx through L-type Ca(2+) channels show that Septin5 depletion increases releasable granule pool clustering on Ca(2+) channels, previously shown to be impaired in T2D, thus rectifying this T2D defect. Hence, inhibition of Septin5 can improve insulin secretion.