Objective To investigate the method to isolate and culture hepatic stellate cells (HSCs) for studying the cellular mechanisms of hepatic frbrosis. Methods HSCs were isolated by nycodenz density gradient centrifugation after the hepatocytes obtained from adult male gebils were digested with pronase, collagenase and DNase, infused via portal vein. The cell viability was determined by trypan blue exclusion test. The purity of HSCs was identified by detecting α-SMA, desmin immunohistochemical staining. Results The yield rate of HSCs was 0.5~1×10⁷ per gerbil liver, and the cell viability was more than 90%. The percentage of α-SMA-positive cells was more than 75% after 3 days primary culture and almost 100% cells were α-SMA and desmin positive in passage culture. Conclusion The successful protocol of primary culture of Mongolian gerbil HSC provide a technical support for research of relevant liver diseases and drug development in the future.