Autophosphorylation of inositol-requiring enzyme 1alpha (IRE1alpha) is required for its activation, which elicits the cellular unfolded protein response (UPR) and is functionally connected with insulin biosynthesis in pancreatic beta cells. We found that the scaffold protein receptor for activated C-kinase 1 (RACK1) interacted with IRE1alpha in a glucose-stimulated or endoplasmic reticulum (ER) stress-responsive manner in pancreatic beta cells and primary islets. RACK1 mediated the glucose-inducible assembly of a complex containing IRE1alpha, RACK1, and protein phosphatase 2A (PP2A) to promote dephosphorylation of IRE1alpha by PP2A, thereby inhibiting glucose-stimulated IRE1alpha activation and attenuating IRE1alpha-dependent increases in insulin production. Moreover, IRE1alpha activation was increased and RACK1 abundance was decreased in a mouse model of diabetes. Thus, our findings demonstrate that RACK1 functions as a key component in regulating the IRE1alpha signaling pathway in pancreatic beta cells.