Objective: To investigate the actions of ROCKI/II in phenotypic transformation of human aortic vascular smooth muscle cells (HA-VSMCs) induced by transforming growth factor β1 (TGF-β1). Methods: HA-VSMCs were respectively transfected with ROCKI and ROCKII, and the transfection results were observed by fluorescence microscope. The ROCKI and ROCKII protein expressions in HA-VSMCs with different treatments (ROCKI siRNA transfection, ROCKII siRNA transfection, +TGF-β1, ROCKI siRNAtransfection+TGF-β1, and ROCKII siRNAtransfection+TGF-β1) were determined by Western blot analysis. The protein and mRNA expressions of the contractile phenotype maker α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α) and synthetic phenotype marker osteopontin (OPN) in HA-VSMCs with different treatments (+TGF-β1, ROCKI siRNA transfection+TGF-β1, ROCKII siRNA transfection+TGF-β1, and pretreatment of ROCK non-specificity Y-27632+TGF-β1) were determined by Western blot analysis and RT-PCR method, respectively. Untreated HA-VSMCs were used as blank control. Results: Both siRNAs were successfully transfected as evidenced by fluorescence observation and Western blot analysis. In HA-VSMCs after TGF-β1 treatment, the ROCKI protein expression level was significantly up-regulated (P<0.05), but the ROCKII protein expression level did not significantly change (P>0.05), while the ROCKI increasing effect of TGF-β1 was significantly inhibited by ROCKI siRNA transfection (P<0.05). In HA-VSMCs after TGF-β1 treatment, the protein and mRNA expressions of α-SMA and SM22α were decreased and those of OPN were increased significantly (all P<0.05), and these effects were significantly suppressed by ROCKI siRNA transfection or Y-27632 pretreatment (all P<0.05), but were not affected by ROCKII siRNA transfection (all P>0.05). Conclusion: TGF-β1 can induce the transformation of HA-VSMCs from contractile phenotype to synthetic phenotype, which may be associated with the up-regulation of ROCKI expression.