Although retinal microvessels (RMVs) and brain microvessels (BMVs) are closely related in their developmental and share similar blood-neural barriers, studies have reported markedly different responses to stressors such as diabetes. Therefore, we hypothesized that RMVs and BMVs will display substantial differences in gene expression levels even though they are of the same embryological origin. In this study, both RMVs and BMVs were mechanically isolated from rats. Full retinal and brain tissue samples (RT, BT) were collected for comparisons. Total RNA extracted from these four groups were processed on Affymetrix rat 2.0 microarray Chips. The transcriptional profiles of these tissues were then analyzed. In the present paper we looked at differentially expressed genes (DEGs) in RMVs (against RT) and BMVs (against BT) using a rather conservative threshold value of >= +/- twofold change and a false discovery rate corrected for multiple comparisons (p twofold enriched in BMVs. Using these DEGs, we comprehensively analyzed the actual expression levels and highlighted their involvement in critical functional structures in RMVs and BMVs, such as junctional complex, transporters and signaling pathways. Our work provides for the first time the transcriptional profiles of rat RMVs and BMVs. These results may help to understand why retina and brain microvasculature show different susceptibilities to stressors, and they might even provide new insight for pharmacological interventions.