Hepatitis E virus (HEV) is considered as an emerging global health problem. The pathogen is responsible for more than 2 000 cases of acute hepatitis in France every year resulting in majority from zoonotic infections associated with the consumption of raw or undercooked pork. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. For decades, the lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin inhibited HEV replication in HepaRG cells. Using whole genome sequencing, 25 mutations including 8 non-synonymous mutations were detected in the genome of the virus recovered after 6 passages into HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to identify host–virus interactions and putative mutations within the viral genome than can occur in vitro in the context of prolonged hepatitis E infection and to test antiviral drugs against chronic HEV infection.