Many diseases of the eye such as retinoblastoma, diabetic retinopathy, and retinopathy of prematurity are associated with blood-retinal barrier (BRB) dysfunction. Identifying the factors that contribute to BRB formation during human eye development and maintenance could provide insights into such diseases. Here we show that A-kinase anchor protein 12 (AKAP12) induces BRB formation by increasing angiopoietin-1 and decreasing vascular endothelial growth factor (VEGF) levels in astrocytes. We reveal that AKAP12 downregulates the level of hypoxia-inducible factor-1alpha (HIF-1alpha) protein by enhancing the interaction of HIF-1alpha with pVHL (von Hippel-Lindau tumor suppressor protein) and PHD2 (prolyl hydroxylase 2). Conditioned media from AKAP12-overexpressing astrocytes induced barriergenesis by upregulating the expression of tight junction proteins in human retina microvascular endothelial cells (HRMECs). Compared with the retina during BRB maturation, AKAP12 expression in retinoblastoma patient tissue was markedly reduced whereas that of VEGF was increased. These findings suggest that AKAP12 may induce BRB formation through antiangiogenesis and barriergenesis in the developing human eye and that defects in this mechanism can lead to a loss of tight junction proteins and contribute to the development of retinal pathologies such as retinoblastoma.