This study aimed at obtaining an in-depth mapping of expressional changes of the cerebral microvasculature after transient global cerebral ischemia (GCI) and the impact on these GCI-induced expressional changes of post-GCI treatment with a mitogen-activated protein kinase kinase (MEK1/2) inhibitor. GCI was induced in male Wistar rats followed by treatment with either vehicle or the MEK1/2 inhibitor U0126 every 12 h post-GCI. Seventy-two hours after GCI or sham surgery, the cerebral microvasculature was isolated and the protein content analysed with state-of-the-art mass spectrometry. The proteomic profile of the isolated cerebral microvasculature 72 h after GCI (compared to sham) indicated that the main expressional changes could be divided into nine categories: (1) cellular respiration, (2) remodelling of the extracellular matrix, (3) decreased contractile phenotype, (4) clathrin-mediated endocytosis, (5) ribosomal activity, (6) expression of chromatin structure-related proteins, (7) altered synaptic activity, (8) altered G-protein signalling and (9) instability of the membrane potential. Treatment with U0126 partly normalized the expression of one or more of the proteins in all nine categories. Flow cytometry confirmed key findings from the proteome such as upregulation of the extracellular proteins lamininβ2 and nidogen2 (p < 0.05) after GCI. These results provide valuable molecular insight into the broad and complex expressional changes in the cerebral microvasculature after GCI and the effect of early MEK1/2 inhibitor treatment on these changes.