Mice, deficient for vascular endothelial growth factor VEGF-A in pancreatic islets, have reduced insulin gene expression levels and an impaired glucose tolerance. Here, we investigated whether VEGF-A was required for physiological glucose-stimulated insulin secretion and insulin content. We performed in situ pancreas perfusions and islet perifusions on mice lacking VEGF-A in the pancreatic epithelium in order to study their ability to secrete insulin in response to glucose. We identified insulin secretion defects in the pancreata of VEGF-A deficient mice, including a delayed and blunted response to glucose. Islet perifusion experiments revealed a missing first phase and weaker second phase of insulin secretion, in two of three VEGF-A deficient mice. On average, insulin content in VEGF-A deficient islets was significantly reduced when compared with control islets. We conclude that VEGF-A is required in pancreatic islets for normal glucose-stimulated insulin secretion and physiological insulin content. Thus, VEGF-A is a key factor for pancreatic islet function.