Increased homocysteine (Hcy) levels contribute to a variety of cardiovascular and cerebrovascular diseases including stroke and Alzheimers disease. Recent data has shown that elevated levels of Hcy can lead to blood-brain barrier (BBB) dysfunction and activation. However, the mechanism for Hcy-mediated dysfunction remains unclear. The aim of this study is to characterize the effects of moderate Hcy administration in rat brain capillary endothelial cells (BCECs), which serve as a simple model to study blood-brain barrier functions. This present study shows that addition of 20 μM Hcy for 6 days does not significantly affect BCEC survival, as measured by acridine orange staining, propidium iodide staining, and nitrite content. However, addition of 20 μM Hcy for 6 days does elevate lactate dehydrogenase (LDH) activity released into the supernatant of BCECs, as well as significantly enhances the transmigration of monocytes across the BCEC in a time-dependent manner. In addition, TNFα levels in BCEC are also elevated by Hcy, whereas inflammatory markers MIP3α and RANTES are significantly reduced. Finally, this study shows that intercellular adhesion molecule-1 (ICAM-1) expression is significantly enhanced by 20 μM Hcy treatment compared to control conditions. These results suggest that moderate levels of homocysteine can affect proinflammatory patterns expressed by BCECs, ultimately leading to BBB activation and dysfunction through enhanced monocyte transmigration and ICAM-1 expression.