To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. At a high concentration, acrolein (100 μM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 μM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.