Aim To clone human Cystatin C gene and construct its eukaryotic expression vector to observe the expression in the human vascular smooth muscle cells (VSMC). Methods Cystatin C gene was obtained from human endothelial cells by reverse transcription-polymerase chain reaction (RT-PCR). Molecular cloning technique was used to construct this kind of eukaryotic expression vector. pCDNA3.1-Cystatin C was identified by restriction enzyme and PCR. Using liposome-mediated transfection, the eukaryotic expression vector pCDNA3.1-Cystatin C was transfected into human VSMC and proved by RT-PCR and Western blot. Results The eukaryotic expression vector pCDNA3.1-Cystatin C was constructed correctly and was highly expressed in human VSMC. Conclusion Cystatin C was cloned successfully, its eukaryotic expression vector was constructed correctly and was expressed highly in human VSMC.