A growing body of evidence has underlined the significance of endoplasmic reticulum (ER) stress in the pathogenesis of diabetes mellitus. ER oxidoreductin 1β (ERO1β) is a pancreas-specific disulfide oxidase that is known to be upregulated in response to ER stress and to promote protein folding in pancreatic β cells. It has recently been demonstrated that ERO1β promotes insulin biogenesis in β cells and thus contributes to physiological glucose homeostasis, though it is unknown if ERO1β is involved in the pathogenesis of diabetes mellitus. Here we show that in diabetic model mice, ERO1β expression is paradoxically decreased in β cells despite the indications of increased ER stress. However, overexpression of ERO1β in β cells led to the upregulation of unfolded protein response genes and markedly enlarged ER lumens, indicating that ERO1β overexpression caused ER stress in the β cells. Insulin contents were decreased in the β cells that overexpressed ERO1β, leading to impaired insulin secretion in response to glucose stimulation. These data indicate the importance of the fine-tuning of the ER redox state, the disturbance of which would compromise the function of β cells in insulin synthesis and thus contribute to the pathogenesis of diabetes mellitus.