Hepatitis C is caused by hepatitis C virus (HCV)infection, and is the leading cause of liver cirrhosis and liver cancer. HCV has become a serious threat to human health and become public health problems. HCV nonstructure protein 5A is a target and hotspot of HCV inhibitor research in recent years. And we reviewed three domains of NS5A and the biological functions of each domain in hepatitis C virus replication, virus particle assembly and release. It provides a broad idea for the study of the mechanism of action of NS5A inhibitors, and is beneficial to the development of NS5A inhibitors.