Differentiated vascular smooth muscle cells (VSMC) are characterized as expression of specific marker genes, elongated and spindle-shaped morphology and the ability to contract in response to agonist stimulation. To determine whether human VSMC phenotype could revert from dedifferentiation to differentiation, over confluent VSMC was incubated in serum-free M199, and expression of differentiation marker genes was detected by RT-PCR, Northern blotting and Western blotting. The relationships between attainment of a contractile phenotype and expression of differentiation marker genes were investigated. The results indicated that over confluent VSMC after serum withdrawal sequentially expressed specific contractile proteins, including SM α-actin, SM-MHC isoforms SM1 and SM2, h1-calponin and SM22α. During prolonged periods of “starvation”, the increases in the expression of marker genes were associated with elevation of SRF binding to CArG cis-elements. On serum withdrawal, VSMC expressed the transcription factors that are necessary for regulating VSMC differentiation, such as SmLIM, Gax and HRG-1, which were restricted in differentiated VSMC expression and undetectable before serum withdrawal. After serum removal, these cells organized to form elongated, dense, multilayered cell bundles, and had the ability to contract in response to agonist stimulation. It was demonstrated that over confluent VSMC could revert from a dedifferentiated state to a contractile, differentiated state, and restore agonist-induced contraction.