Objective To study the influence of different culture conditions EM>in vitro/EM> on phenotype, proliferation and cytoskeletal proteins expression of vascular smooth muscle cells(VSMCs). Methods The cultured VSMCs from rat aorta were divided into six groups: P2 control,P2 starvation,P4 control,P4 starvation,P6 control and P6 starvation. The proliferating cells were labeled by 5bromodeoxyuridine (5-BrdU); The mRNA expression of smooth muscle 22 alpha (SM22α) was detected by reverse transcriptionpolymerase chain reaction method (RT-PCR); The cytoskeletal proteins including SMα-actin,β-Tubulin and Desmin were observed through immunohistochemical staining, Results With the increase of cell passage, cytoskeletal proteins expression of VSMCs decreased,cellular organs increased and secretory vesicles were abundant; in serumfree cultured cells mitochondria increased and electron density enhanced in cytoplasm of VSMCsOn the contrary the expression of SMα-actin decreased, and the expression of SMα-actin increased, The expression of β-Tublin and Desmin decreased more obviously, and at 6 passages failed to express. Conclusion The conditioned medium and serumfree had the different effects on the phenotype,proliferation and cytoskeleton of VSMCs in different passage, and there was