Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-β1 (TGF-β1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-β1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule-1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-β1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain.