Purpose: Vascular endothelial growth factor (VEGF) is the most potent angiogenic mitogen, and has been associated with angiogenesis. Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, which can induce VEGF expression. The aims of the present study were to evaluate the heparanase expression and its relationship with VEGF in the retina of oxygen-induced retinopathy (OIR) mice, and to investigate the effect of the heparanase inhibitor phosphomannopentaose sulfate (PI-88) in the OIR retinas. Methods: Seventy-seven newborn C57BL/6 mice were involved in this study. On postnatal day 7 (P7), pups were exposed to a hyperoxia condition (75% oxygen) for 5 days, and on P12, the mice were returned to room air. Control mice were exposed to room air from birth until P17, with normally developing retinal vasculature. PI-88 was administered intraperitoneally to OIR mice at a dose of 35.7 mg/kg/day for 5 consecutive days. The expression level of heparanase and VEGF in the retinas was assayed using immunohistochemistry, Q-RT-PCR, and western blot. Results: The expression levels of heparanase and VEGF were increased in the OIR retinas compared with the control mice. The Q-RT-PCR results showed that the mRNA expression levels of heparanase and VEGF in OIR retina were increased 1.71 fold (p<0.0001) and 4.34 fold (p<0.0001), respectively. The western blot results showed that the protein expression levels of heparanase and VEGF were increased 1.49 fold (p<0.0001) and 1.72 fold (p<0.0001), respectively, in the OIR retinas compared with the normal retinas. The immunohistochemistry analysis revealed that the heparanase and VEGF signals were intense in the retinal vascular endothelia of the OIR mice but faint in those of the normal controls. The increased protein and mRNA expression levels of heparanase and VEGF in the mouse retinas were significantly decreased by PI-88 administration (p<0.0001). Conclusions: Heparanase expression was upregulated and correlated with an increase in VEGF expression in the OIR mouse retinas, and might be involved in the progress of retinopathy of prematurity. Inhibition of heparanase expression by PI-88 could be used as a novel therapeutic method for retinopathy of prematurity.