In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.