Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. Although it is clear that tumor necrosis factor-α is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase Toll-like receptor 4 (TLR4) signaling, which could be inhibited by a β-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate β-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (RECs) and rat retinal Müller cells (rMC-1) exposed to high glucose (25 mM), were treated with a novel β-adrenergic receptor agonist, Compound 49b (50 nM), or phosphate-buffered saline (control). TLR4 and its downstream signaling partners (MyD88, IL-1 receptor-associated kinase 1, TNF receptor-associated factor 6 and total and phosphorylated nuclear factor-κB) were examined. In addition, we assessed high-mobility group box 1 (HMGB1) protein levels. Our data showed that diabetes or high-glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in RECs and Müller cells grown in high-glucose culture conditions, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway, providing a potential mechanism for its protective actions.