AbstractPrimary defects in folding of mutant proinsulin can cause dominant‐negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β‐cell dysfunction. Conversely, if primary impairment of ER‐to‐Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin—this might suggest bi‐directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged β‐cells with conditions that impair ER‐to‐Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS‐PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post‐Golgi compartments results in intracellular accumulation of properly‐folded proinsulin (bearing native disulfide bonds), impairment of ER‐to‐Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER‐to‐Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre‐existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER‐to‐Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.