Aim To investigate the effect of culture of vascular smooth muscle cell (VSMC)in vitro on its phenotype transformation and to survey the best parameters for characterizing the phenotype. Methods The primary VSMC and the fourth passage of VSMC in vitro culture were compared by their morphological characteristics under electron microscope, and the α-SM actin expression measured by immunohistochemical staining, the osteopontin (OPN), SM22α, matrix Gla protein (MGP) mRNA transcription levels measured by RT-PCR were all comparatively studied. Results The myofilament was abundant, the dense patch—a typical structure of contractile phenotype of VSMC well-distributed, while the endoplasmic reticulum, Golgi's complex were less-distributed in primary VSMC. The VSMC in fourth passage, however, showed scattered distribution of myofilament in cytosol, but much more endoplasmic reticulum, Golgi's complex, and mitochondria under electron microscope observation. The immunohistochemical staining showed much strongerα-SM actin expression in primary VSMC compared with the fourth passage (P<0.05). OPN mRNA level increased very significantly (P<0.01) in fourth passage of VSMC compared with primary ones. The transcription level of SM22α mRNA in fourth passage of VSMC decreased compared with primary VSMC, but the difference was not significant (P>0.05). MGP mRNA transcription levels increased in fourth passage of VSMC (P<0.05). Conclusions Four passages of VSMC in vitro can transform the typical contractile phenotype into the typical synthetic phenotype, which indicated that the VSMC had the trait of easy-dedifferentiation, this may suggest some implication in VSMC research. Electron microscope observation, α-SM actin, OPN showed significant difference between contractile phenotype and synthetic phenotype, which may represent a good marker for distinguishing the two different phenotypes. The SM22α and the matrix Gla had some difference, but overlaps were also significant between the two different phenotypes.