Objective To clarify the impairment mechanisms of acute hyperglycemia in the first-phase insulin secretion in mice. Methods The mouse model of acute glucose toxicity was established by glucose infusion through jugular vein catheterization. The glucose and insulin levels were assessed by IPGTT and OGTT in the mice of acute hyperglycemia and control groups. The histology of pancreatic islets was observed using HE staining and the insulin granules and other cytoplasmic organelles were observed by electron microscopy.Results The mouse model of acute hyperglycemia was successfully established. The IPGTT showed that the blood glucose level was decreased by 87% (10.3±0.33 mmol/L vs. 19.3±1.66 mmol/L) at 15 min in the acute hyperglycemia group compared with the control group. The OGTT showed that the blood glucose level was decreased by 85% (9.8±0.31 mmol/L vs. 18.16±1.01 mmol/L) at 30 min in the acute hyperglycemia group compared with the control group. However, the peak values of insulin secretion were delayed in both IPGTT and OGTT. Insulin levels at 2.8 and 16.7 mmol/L glucose stimulation in the acute hyperglycemia group was declined by 46% and 67% than the control group, respectively (P<0.05). Residual insulin content in islet β cells was declined by 49% at 2.8 mmol/L and 94% at 16.7 mmol/L glucose infusion than the control group (P<0.05). The histology showed irregular structure of pancreatic islets in the acute hyperglycemia group. The electron microscopy revealed that the amount of insulin granules was decreased, and more cytoplasmic vacuoles and swollen mitochondria were observed.Conclusions Acute intravenous glucose load decreases insulin content of islet β cells, leading to decrease and delay of the first-phase insulin secretion.