Hyperglycemia increases insulin flux through the endoplasmic reticulum (ER) of pancreatic β-cells, and the unfolded protein response pathway is required to enhance insulin processing. Pancreatic and duodenal homeobox 1 (PDX1), a key pancreatic transcription factor, regulates insulin along with targets involved in insulin processing and secretion. Here we find that PDX1 is a direct transcriptional regulator of ER oxidoreductin-1-like β (Ero1lβ), which maintains the oxidative environment of the ER to facilitate disulfide bond formation. PDX1 deficiency reduced Ero1lβ transcript levels in mouse islets and mouse insulinoma (MIN6) cells; moreover, PDX1 occupied the Ero1lβ promoter in β-cells. ERO1lβ levels were induced by high glucose concentrations and by the reducing agent dithiothreitol, indicating potential roles in adaptation to increased oxidative protein folding load in the β-cell ER. In MIN6 cells, small interfering RNA-mediated silencing of Ero1lβ decreased insulin content and increased susceptibility to ER stress-induced apoptosis. These findings demonstrate roles for the PDX1 target ERO1lβ in maintaining insulin content and regulating cell survival during ER stress.