Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin's regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes (BRPC), retinal endothelial cells and retinal pigment epithelial cells. In BRPC, insulin induced mRNA and protein expression of HO-1 in a time and concentration dependent manner. Using HO-1 promoter analysis, luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in ho-1 promoter gene which was not responsive to antioxidants. Insulin induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2 and Akt dominant negative or siRNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H2O2-induced NF-B and caspase-8 activation and apoptosis via IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that insulin's anti-apoptotic properties are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation by which insulin is retarding the progression of microvascular complications induced by diabetes.