We have recently reported that Compound 49b, a novel β-adrenergic receptor agonist, can significantly reduce VEGF levels in retinal endothelial cells (REC) grown in diabetic-like conditions. In this study, we investigated whether Compound 49b could protect the retina under hypoxic conditions using the ischemia-reperfusion (I/R)-induced model in rats, as well REC cultured in hypoxic conditions. Some rats received 1mM topical Compound 49b for the 2 (5 rats each group) or 10 (4 rats in each group) days post-I/R. Analyses for retinal thickness and cell loss in the ganglion cell layer was done at 2 days post-I/R, while numbers of degenerate capillaries and pericyte ghosts were measured at 10 days post-I/R. Additionally, REC were cultured in normal oxygen or hypoxia (5% O2) only or treated with 50 nM Compound 49b for 12 hours. Twelve hours after Compound 49b exposure, cells were collected and analyzed for protein levels of insulin-like growth factor binding protein 3 (IGFBP-3), vascular endothelial cell growth factor (VEGF) and its receptor (KDR), angiopoietin 1 and its receptor Tie2 for Western blotting. Data indicate that exposure to I/R significantly decreased retinal thickness, with increasing numbers of degenerate capillaries and pericyte ghosts. Compound 49b treatment inhibited these retinal changes. In REC cultured in hypoxia, levels of IGFBP-3 were reduced, which were significantly increased by Compound 49b. Hypoxia significantly increased protein levels of VEGF, KDR, Angiopoiein 1, and Tie2, which were reduced following Compound 49b treatment. These data strongly suggested that Compound 49b protected the retina against I/R-induced injury. This provides additional support for a role of β-adrenergic receptor actions in the retina.