In vitro experiments and in vivo studies on rodents demonstrate that N-terminal 14, 15, 16, 17, and 18 kDa fragments prolactin-related vasoinhibin (PRL-V) of human PRL are natural inhibitors of neovascularization in the retina and elsewhere. These N-terminal PRL fragments belong to a family of peptides named vasoinhibins, which act as endogenous regulators of angiogenesis and vascular function. These observations led to the hypothesis that PRL-V could play a role in the pathophysiology of diabetic retinopathy in humans. The purpose of this study was to investigate whether patients with diabetes mellitus and diabetic retinopathy have aberrant concentrations of PRL-V in the circulating blood. We performed a case-control study and developed a new technique to semi-quantitatively determine PRL-V in serum samples from 48 male subjects. The case group consisted of 21 patients with diabetes mellitus and proliferative or non-proliferative diabetic retinopathy. The control group consisted of 27 healthy subjects with no history of diabetes mellitus. For the detection of PRL-V, we developed a new analytical method, consisting of immunologic and laser-induced fluorescence techniques. Results The case group had significantly lower PRL-V serum concentrations than the control group (P=0.041). There was no significant difference between patients with proliferative and those with non-proliferative diabetic retinopathy. We conclude that given the antiangiogenic and antivasopermeability actions of PRL-V, the decreased serum levels of PRL-V in patients with diabetes mellitus could contribute to the development and progression of diabetic retinopathy.