Endothelial cells (ECs) line the innermost of the blood vessel wall and are constantly subjected to shear stress imposed by blood flow. ECs were also influenced by the neighboring vascular smooth muscle cells (VSMCs). The bidirectional communication between ECs and VSMCs modulates vascular homeostasis. In this study, the involvement of histone deacetylase 6 (HDAC6) in modulating migration of ECs co-cultured with VSMCs by the normal level of laminar shear stress (NSS) was investigated. ECs was either cultured alone or co-cultured with VSMCs under static conditions or subjected to NSS of 15 dyne/cm2 by using a parallel-plate co-culture flow chamber system. It was demonstrated that both NSS and VSMCs could increase EC migration. The migration level of ECs co-cultured with VSMCs under NSS was not higher than that under the static condition. The process of EC migration regulated by VSMCs and NSS was associated with the increased expression of HDAC6 and low level of acetylated tubulin. The increase in HDAC6 expression was accompanied by a time-dependent decrease in the acetylation of tubulin in ECs co-cultured with VSMCs. Inhibition of the HDAC6 by siRNA or tributyrin, an inhibitor of HDACs, induced a parallel alteration in the migration and the acetylated tubulin of ECs co-cultured with VSMCs. It was observed by immunofluorescence staining that the acetylated tubulin was distributed mostly around the cell nucleus in ECs co-cultured with VSMCs. The results suggest that the NSS may display a protective function on the vascular homeostasis by modulating EC migration to a normal level in a VSMC-dependent manner. This modulation process involves the down-regulation of acetylated tubulin which results from increased HDAC6 activity in ECs.