Abstract: Vascular smooth muscle cells (VSMCs) are crucial for studying the mechanism and regulation of vasogenic response, but so far no ideal method has been reported for their isolation. We established a VMSC isolation method based on two-step digestion, with which a large population of single smooth muscle cells could be obtained. After cell isolation, the potassium current, cytosolic calcium concentration, reactivity to caffeine were measured, and the results demonstrated that the cells were highly viable and exhibited reactivity to the vascular constrictor to meet various needs in experimental research.