Objective The influence of shear stress on the assembly of microtubules in endothelial cells (ECs) was elucidated in the co-culture system with vascular smooth muscle cells to provide some experimental evidences for molecular mechanisms of shear stress-induced vascular remodeling. Methods The assembly of microtubules in ECs co-cultured with vascular smooth muscle cells (VSMCs) under shear stressof 15dyne/cm2- was examined by Western Blot, immunocytochemistry and image analysis. ECs were co-cultured with VSMCs under static conditions as a control. Results The microtubule arrangements in the control group showed sparsely, emanative and ruleless. After laminar shear stress (1.5Pa, 15dynes/cm2) applied to ECs co-cultured with VSMCs for 12h, a distinct change in microtubule arrangement appeared to the regular state and oriented to the direction of shear stress. Application of shear stress to the co-cultured ECs also promoted the quantitative assembly of microtubules in ECs. The polymeric tubulin mass became decreased in ECs after exposed to shear stress for 3 hours. Conclusions Shear stress induces and promotes microtubule assembly in the co-cultured ECs. Furthermore, these data suggest that microtubules could be a potential target for translating changes in externally applied mechanical stimulation to alterations in cellular phenotype and functions such as adhesion and migration.