Proliferation and migration of vascular smooth muscle cells (VSMCs) are believed to develop atherosclerosis and venous bypass graft disease. Ligustilide is widely used to treat some pathological settings such as atherosclerosis and hypertension. The aim of this study was to examine the effect of ligustilide on VSMCs proliferation. The results show that ligustilide significantly inhibited VSMCs proliferation and cell cycle progression. Further analysis shows that ligustilide suppressed reactive oxygen species production and extracellular signal-related kinases (ERK), c-Jun N-terminal protein kinase (JNK), and p38 MAP kinase. Cells were treated with antioxidant, superoxide dismutase, catalase, and DPI, respectively, leading to repress ERK, JNK, and p38 activation. The inhibitors of mitogen activated protein kinase (MAPK), PD98059, SB203580, and Sp600125, inhibited cell proliferation. These findings suggest the antiproliferative effect of ligustilide was associated with the decrement of reactive oxygen species resulting in the suppression of MAPK pathway. Thus, ligustilide contribute to be the effective agent in preventing cardiovascular diseases.