Objective To investigate the effect of glucose transporter-1(GLUT1) suppression on microvasculature pathological changes in diabetic mice. Methods Thirty-six 8-week-old C57BL/6 mice were randomly divided into normal control, diabetic control and GLUT1siRNA treatment groups. Diabetic model was established by intraperitoneal injection of streptozotocin. GLUT1siRNA treatment group received intravitreal injection of siRNA-mediated GLUT1, and the other two groups received equal amount of non-specific siRNA. Twenty-one weeks after diabetic induction, immunoblotting was conducted to examine the expression of GLUT1 and two inflammation factors: ICAM-1 and TNF-α. We also calculated the retinal glucose concentration. Leukostasis assay and vascular leakage assay were utilized to compare the microvasculature pathological changes between the two groups. Results The expression of GLUT1 was significantly down-regulated in GLUT1siRNA treatment group compared with the other two groups (P<0.01), decreased by 77.00% compared with the normal control group and only being 8.07% of the diabetic control group. Though retinal glucose concentration of diabetic control and GLUT1siRNA treatment groups were higher than that of normal control, that in GLUT1siRNA treatment group was only 50.05% that of the diabetic control group (P<0.01). The expressions of ICAM-1 and TNF-α in GLUT1siRNA treatment and diabetic control were significantly higher than that in the normal control group, and their expressions in GLUT1siRNA treatment group were 66.14% (P<0.05)and 54.76% (P<0.01)those of the diabetic control group, respectively. Moreover, compared with GLUT1siRNA treatment group, diabetic control group had more adherent leukocytes in the retinal microvasculature and larger areas of leaked FITC-labeled albumin retina. Conclusion GLUT1siRNA can limit glucose transport into the retina by inhibiting GLUT1 expression, thus decrease retinal glucose concentration and alleviate microvasculature changes of diabetic retinopathy.