IRE1α plays an important role in the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. 4μ8C, a well-known inhibitor of IRE1α RNase activity, is commonly used to analyze IRE1α function during ER stress in cultured mammalian cells. However, the off-target effects of 4μ8C remain elusive. Pancreatic β-cells synthesize a large amount of insulin in response to high glucose stimulation, and IRE1α plays an important role in insulin secretion from pancreatic β-cells. Here, to analyze the role of IRE1α in pancreatic β-cells, we examined insulin secretion after 4μ8C treatment. Although 4μ8C inhibited insulin secretion within 2 hr, neither insulin synthesis nor maturation was inhibited by 4μ8C under the same conditions. This result prompted us to examine the precise effects of 4μ8C on insulin secretion in pancreatic β-cells. Unexpectedly, with just 5 min of treatment, 4μ8C blocked insulin secretion in cultured pancreatic β-cells as well as in pancreatic islets. Furthermore, insulin secretion was prevented by 4μ8C, even in pancreatic β-cells lacking the IRE1α RNase domain, suggesting that 4μ8C blocked the late stage of the insulin secretory process, independent of the IRE1α-XBP1 pathway. Our results indicate that 4μ8C has an off-target effect on insulin secretion in pancreatic β-cells. These finding inform the researchers in the field that the use of 4μ8C requires the special consideration for the future studies.