To investigate the mechanism of the cellular repressor of E1A-stimulated genes (CREG) on the differentiation of vascular smooth muscle cells (VSMCs), the human internal thoracic artery cells (named HITASY cells) were stably infected with sense-CREG [pLNCX₂(＋)/CREG] and antisense-CREG [pLXSN(－)/CREG] retroviral vectors and positive clone was abtained by G418 selection. The cells infected with pLNCX₂(＋)/CREG take on the differentiated phenotype and overexpression of CREG can enhance SM α-actin expression accompanied with increase of the total RhoA and nuclear serum response factor (SRF) expression detected by immunofluorescence and Western blot. And treated with a selective Rho kinase inhibitor Y-27632, the expression of SM α-actin and nuclear SRF protein induced by CREG was effectively reduced. Meanwhile, the opposite results were observed in the CREG-depression HITASY cells. And immunoprecipitation assays indicate that as a secreted protein, CREG can be detected in the media of pLNCX₂(＋)/CREG cells and can interact with insulin-like growth factorⅡ/mannose 6-phosphate receptor (M6P/IGF2R). Furthermore, that using okadaic acid (an inhibitor of the protein phosphatases 2A) to decrease the number of M6P/IGF2R at the cell surface can significantly inhibit the expression of total RhoA, nuclear SRF and SM α-actin induced by CREG-overexpression. Taken together, as a secreted protein, CREG can enhance the expression of SM α-actin by interaction with M6P/IGF2R and perform a pivotal role in the process of VSMCs differentiation and phenotype modulation.