In pancreatic β cells, the endoplasmic reticulum (ER) is an important site for insulin biosynthesis and the folding of newly synthesized proinsulin. Here, we show that IRE1α, an ER-resident protein kinase, has a crucial function in insulin biosynthesis. IRE1α phosphorylation is coupled to insulin biosynthesis in response to transient exposure to high glucose; inactivation of IRE1α signaling by siRNA or inhibition of IRE1α phosphorylation hinders insulin biosynthesis. IRE1 activation by high glucose does not accompany XBP-1 splicing and BiP dissociation but upregulates its target genes such as WFS1. Thus, IRE1 signaling activated by transient exposure to high glucose uses a unique subset of downstream components and has a beneficial effect on pancreatic β cells. In contrast, chronic exposure of β cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene expression. IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis.