Introduction: Diabetic retinopathy (DR) is a major complication of diabetes. Anti-Vascular Endothelial Growth Factor (VEGF) drugs represent the standard of care for DR; however, lack of response occurs in approximately 50% of patients. Therefore, a better understanding of DR pathogenesis is required to identify new therapeutic targets. Methods: Vitreous samples were collected from anti-VEGF non-responder (non-RES) patients; non-RES heat-inactivated (HI) vitreous were used as control. To develop a new orthotopic model of DR, non-RES or HI vitreous were injected intravitreally in mice and after 48 hours retinas were explanted and analyzed. The composition of non-RES vitreous was assessed by an angio-inflammatory protein array. The effect of the inhibition of the bioactive heparin-binding proteins (HBPs) present in non-RES vitreous was evaluated in the endothelial spheroid sprouting assay. Results: Orthotopic injection of non-RES vitreous upregulated pro-angiogenic/pro-inflammatory mediators; moreover, it induced alterations of retinal vessels, accumulation of inflammatory infiltrate, and activation of astrocyte gliosis. Notably, HI vitreous was ineffective, pointing to a proteinaceous nature of the non-RES vitreal mediators acting on mouse retinae. Indeed, the blockade of vitreal HBPs mediated by the biotechnological heparin-like molecule K5-N, OS(H) or by the pentapeptide Boc2, completely abrogated the activity of non-RES samples. It is worth noticing that the potency of anti-HBP compounds was significantly higher than the anti-VEGF ranibizumab. Conclusions: Our data show that VEGF is only part of a complex machinery regulating angiogenesis, inflammation and edema formation during DR and that drug resistance is due to the activation of compensatory mechanisms mediated by HBPs besides VEGF.