Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.