Background and Objectives: The Blood-Brain Barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells (ECs). Numerous studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. This has led us to hypothesize that shear stress up-regulates BBB function via direct modulation of the expression and properties of the pivotal tight junction proteins, occludin and ZO-1. Methods & Results: Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady (non-pulsatile) or pulsatile shear stress (10 and 14 dynes cm^-2, respectively) for 24 h. Sheared BBMvECs were then monitored for occludin/ZO-1 expression, association (co-immunoprecipitation) and subcellular localization. Transendothelial permeability of BBMvECs to FITC-dextran and ¹⁴[C]-sucrose was also assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Significant increases in occludin mRNA and protein expression (2.73±0.26 and 1.83±0.03 fold, respectively;n=3) and in occludin/ZO-1 association (2.12±0.15 fold; n=5) were also observed. Moreover, steady shear stress also induced a clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein expression of both occludin and ZO-1 (2.15±0.02 and 1.67±0.21 fold, respectively; n=3) and increased occludin/ZO-1 association (2.91±0.14 fold; n=3) were observed in parallel with a reduction in transendothelial permeability to ¹⁴[C]-sucrose by 19.7±7.74%. Conclusions: Shear stress up-regulates BBMvEC barrier function at the molecular level via modulation of the expression, association and localization of occludin and ZO-1. Moreover, the pulsatile shear model appeared to give the most profound biochemical responses.