The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.