Epoxyeicosatrienoic acids (EETs) are generated by the cytochrome P450 (CYP) from arachidonic acid and mainly metabolized by the soluble epoxide hydroylase (sEH). We investigated the role of CYP/sEH pathway in the regulation of vascularization in the retina. Retinal angiogenesis was investigated at different time points after birth. Vascularization was markedly delayed in sEH^-/- mice at postnatal day (P) 2 and P5, and associated with reduced tip cell numbers, filopodia extension and attenuated endothelial cell proliferation. This phenotype could be reproduced in wildtype mice by giving sEH inhibitors. The formation of deeper capillaries was also significantly suppressed in sEH^-/- at P9. We found that retinal sEH is highly expressed in astrocytes and Müller cells. Therefore we created an astrocyte as well as Müller cell specific sEH knockout mice for further investigation. The astrocyte specific sEH knockout mice exhibited a significant reduction in vessel density at P5 and P7. Whereas Müller cell specific sEH knockout mice showed a reduced vessel radial expansion at P2 and P5. Moreover, conditioned medium from wildtype Müller cells was able to stimulate significantly more endothelial cell proliferation compared to sEH^-/-; the proliferation was abolished by a VEGF neutralizing antibody. qRT-PCR analysis showed an consistent induction of Notch dependent transcription factors Hes1 and Hey1 in sEH^-/- mice. These data indicate that the CYP/sEH cascade is required for retinal vascularization. This involves a sEH-regulated release of angiogenic factors from glia cells which activates the Notch pathway in endothelial cells.