Transplantation of pancreatic islets is clinically used to treat type 1 diabetes but requires multiple donors. Previous experimental studies demonstrated that transplanted islets have a low blood vessel density, which leads to a hypoxic microenvironment. The present study tested the hypothesis that experimental prolactin pretreatment, a substance that seems to stimulate angiogenesis in endogenous islets, would increase graft blood vessel density, thereby improving transplantation outcome. Pancreatic islets from C57BL/6 mice were incubated with prolactin (500 ng/ml) or vehicle during the last 24 h of culture before syngeneic transplantation beneath the renal capsule, or recipients were injected with prolactin or vehicle for the first 7 d after transplantation. One month after transplantation, graft vascular density, blood flow, oxygen tension, endocrine volume, and function were evaluated. Also, human islets were incubated with prolactin or vehicle before experimental transplantation and investigated for vascular engraftment. Vascular engraftment of syngeneically transplanted mouse islets was improved by both in vivo and in vitro prolactin pretreatment. Moreover, prolactin pretreatment in vitro of islets used for transplantation improved recovery from diabetes in a minimal islet mass model. Interestingly, also human islets subjected to prolactin treatment before experimental transplantation demonstrated improved revascularization, blood perfusion, and oxygen tension when evaluated 1 month after transplantation. We conclude that prolactin may improve engraftment of transplanted pancreatic islets. The protocol with pretreatment of islets ex vivo could minimize the risk of side effects when used in the clinical setting.