This study investigated the effects of streptozotocin-induced diabetes on the functional integrity of the blood-brain barrier in the rat at 7, 28, 56, and 90 d using vascular space markers ranging in size from 342 to 65,000 Da. We also examined the effect of insulin treatment of diabetes on the formation and progression of cerebral microvascular damage and determined whether observed functional changes occurred globally throughout the brain or within specific brain regions. Results demonstrate that streptozotocin-induced diabetes produced a progressive increase in blood-brain barrier permeability to small molecules from 28 to 90 d and these changes in blood-brain barrier permeability were region specific with the midbrain most susceptible to diabetes-induced microvascular damage. In addition, results showed that insulin treatment of diabetes attenuated BBB disruption, especially during the first few weeks; however, as diabetes progressed, it was evident that microvascular damage occurred even when hyperglycemia was controlled. Overall, results of this study suggest that diabetes-induced perturbations to cerebral microvessels may disrupt homeostasis and contribute to long-term cognitive and functional deficits of the central nervous system.