Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to phosphatidic acid. We have previously demonstrated that DGKδ acts as a negative regulator of β-cell proliferation. However, it is still unknown whether DGKδ deficiency in pancreatic β-cells alleviates diabetes. In the present study, we investigated the effect of β-cell specific DGKδ deficiency on hyperglycemia in streptozotocin (STZ)-induced diabetic mice. We administered STZ to 5-wk-old mice and performed measurement of body weight and blood glucose level for 60 days. At 11 days after STZ administration, there was no significant difference in serum insulin concentrations, pancreatic insulin content, or β-cell area between β-cell specific DGKδ knockout (βDGKδKO) and control mice. At 60 days, in contrast, blood glucose level was significantly lower and serum insulin level and pancreatic insulin content were significantly higher in βDGKδKO mice than in control mice. In morphological analysis, a significant increase in β-cell area was observed in βDGKδheteroKO mice compared with control mice. These results suggest that DGKδ deficiency in pancreatic β-cells prevents STZ-induced hyperglycemia, which is likely due to maintained β-cell mass resulting from promoted β-cell proliferation.