Objective To establish a method for culture of human mesenteric artery smooth muscle cell (hVSMC) by tissue-piece inoculation in vitro and to provide new technology to study cardiovascular diseases. Methods The tissue-piece inoculation method was applied to cell primary culture of hVSMC. Morphology of the cells was observed by inverted microscopy and identified by immunohistochemical methods. Results A great quantity of single hVSMC were obtained by tissue-piece inoculation. The cultured hVSMC showed typical “peak and valley” pattern and immunohistochemical staining showed that all the cells were positive. Conclusions The hVSMC are obtained by tissue-piece inoculation. This method provides good study material to research cardiovascular diseases.