The pancreatic beta cells are not only the fundamental source of insulin production but also the predominant regulator of its storage and release upon appropriate stimuli to maintain glucose homeostasis. Insulin is stored in membrane-bounded structures called secretory granules (SGs), which are specialized secretory units of the regulated secretory pathway. Synthesized proinsulin as an inactive precursor form transports into the Golgi apparatus, where proinsulin is sorted and packaged into immature SGs. Soluble cargo proteins, membrane proteins, as well as ions are critical components for insulin production as well as insulin storage in pancreatic beta cells. Type 2 diabetes (T2D) is a prevalent and complex disease that is defined by chronic hyperglycemia and insulin resistance, which are strongly correlated with dysregulated insulin production machinery in beta cells. Recently, vacuolar protein sorting-associated protein 41 (VPS41) was identified as a regulator in the trafficking pathways of synthesized secretory proteins. In this thesis, I show that VPS41 plays a critical role in insulin storage capacity and insulin granule biogenesis in beta cells using both an in vitro rat insulinoma beta cell VPS41 knockout line and an in vivo mouse model with a conditional deletion of VPS41 in beta cells. I show for the first time that deletion of VPS41 in a mouse model leads to severe diabetes associated with extensive depletion of insulin in pancreatic beta cells. Together, my in vivo and in vitro data illustrate that VPS41 is a potent regulator of insulin secretory biology and is required for the maintenance of normal glucose homeostasis