Objective This paper introduces one modified digestion method with the compound enzymes to prepare the rat aortic vascular smooth muscle cells (VSMC).Methods The collagenase, trypsin inhibitor and elastase are combined together to firstly digest the surgically excised thoracic aorta for the removal of its intima and advantitia. Its separated media layer is then digested again with the same complex enzymes for the release of the aortic VSMCs.Results The isolated VSMC are cultured in vitro,grow into the elongated spindle shaped, and reach the confluency within a week displaying the obvious "hill and valley" pattern. More than 95% of the cells are alpha smooth muscle actin and Myosin-11 positive, and express the typical contractile VSMC marker genes at both the RNA and protein levels. Conclusion This preparative method is pretty simple, easy and reproducible, providing the good yield of the viable and pure contractile aortic VSMC in a short time.