Aim To culture rat vascular smooth muscle cells by novel enzymatic dispersion method. Methods Rat aorta was removed from left subclavian origin to diaphragmatic insertion in sterile conditions. The vascular media was digested,then the cells were centrifuged and plated down in prepared flask. Results The primary vascular smooth muscle cells of rat aorta was performed successfully by using enzymatic dispersion method. The cell viability of third generation is 96% by typan blue,and the cell purity is 97% tested by light microscope and immunocytochemical stains. Conclusions The growth period of enzyme-dispersed vascular smooth muscle cells is shorter by 4～5 days than that of explant method and enzymatic dispersion method reported in domesticity.