Proinsulin is synthesized in the endoplasmic reticulum (ER) in pancreatic β cells and transported to the Golgi apparatus for proper processing and secretion into plasma. Defects in insulin biogenesis may cause diabetes. However, the underlying mechanisms for proinsulin transport are still not fully understood. We show that β cell–specific deletion of cTAGE5, also known as Mea6, leads to increased ER stress, reduced insulin biogenesis in the pancreas, and severe glucose intolerance in mice. We reveal that cTAGE5/MEA6 interacts with vesicle membrane soluble N-ethyl-maleimide sensitive factor attachment protein receptor Sec22b. Sec22b and its interaction with cTAGE5/MEA6 are essential for proinsulin processing. cTAGE5/MEA6 may coordinate with Sec22b to control the release of COPII vesicles from the ER, and thereby the ER-to-Golgi trafficking of proinsulin. Importantly, transgenic expression of human cTAGE5/MEA6 in β cells can rescue not only the defect in islet structure, but also dysfunctional insulin biogenesis and glucose intolerance on cTAGE5/Mea6 conditional knockout background. Together our data provide more insight into the underlying mechanism of the proinsulin trafficking pathway.