α1-Acid glycoprotein (AGP) is a positive acute phase protein which is elevated 1-10 times during inflammation. Whereas AGP has been reported to have immunomodulatory properties, other biological functions of this protein such as its effects on the blood-brain barrier (BBB) endothelium are unknown. Tight junction (TJ) proteins (ZO-1 and occludin) are crucial in maintaining BBB integrity and brain homeostasis. As inflammatory cytokines have been shown to alter BBB integrity and TJ protein expression, we hypothesized that AGP changes BBB function by stimulating inflammatory cytokines and/or directly modulating TJ protein expression. We used primary rat brain microvessel endothelial cells (RBMECs) as an in vitro BBB model to study the direct effects of AGP on the brain microvasculature. No change in cytokine levels was detected in supernatant from AGP-treated RBMECs, despite increased mRNA expression by the cells. Paracellular permeability was decreased up to 20%, across RBMEC monolayers following treatment with AGP, suggesting its role in enhancing BBB integrity. RBMECs showed a biphasic response of increased occludin protein expression following AGP treatment while ZO-1 expression changed in a dose- and time-dependent manner. These changes in TJ proteins suggest that AGP induced changes in occludin related to enhanced barrier properties while the change in ZO-1 may play a secondary role in BBB integrity and/or as an intracellular signaling molecule. AGP significantly changed transcription factor activator protein 1 (AP-1) DNA-binding activity which provides evidence of the potential cell signaling pathways that contribute to the effect of AGP in RBMECs. Together, this supports our hypothesis that AGP has a direct effect in brain microvasculature and may play an important role in altering BBB integrity in inflammatory-related diseases.