High-mobility group box 1 (HMGB1) is a protein that is part of a larger family of non-histone nuclear proteins. HMGB1 is a ubiquitary protein with different isoforms, linked to numerous physiological and pathological pathways. HMGB1 is involved in cytokine and chemokine release, leukocyte activation and migration, tumorigenesis, neoangiogenesis, and the activation of several inflammatory pathways. HMGB1 is, in fact, responsible for the trigger, among others, of nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), toll-like receptor-4 (TLR-4), and vascular endothelial growth factor (VEGF) pathways. Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM) that is rapidly growing in number. DR is an inflammatory disease caused by hyperglycemia, which determines the accumulation of oxidative stress and cell damage, which ultimately leads to hypoxia and neovascularization. Recent evidence has shown that hyperglycemia is responsible for the hyperexpression of HMGB1. This protein activates numerous pathways that cause the development of DR, and HMGB1 levels are constantly increased in diabetic retinas in both proliferative and non-proliferative stages of the disease. Several molecules, such as glycyrrhizin (GA), have proven effective in reducing diabetic damage to the retina through the inhibition of HMGB1. The main focus of this review is the growing amount of evidence linking HMGB1 and DR as well as the new therapeutic strategies involving this protein.