Hepatitis E virus (HEV) causes hepatitis which can resolve by itself or can evolve to fulminant or even chronic hepatitis. For decades, the lack of a robust cell culture system has delayed our knowledge on HEV. Among available cell lines to study HEV, HepaRG can be differentiated with DMSO into hepatocytes and cholangiocytes. Usually, DMSO is maintained upon culture to keep cells differentiated and for the expression of specific proteins. In this work, we addressed the impact of DMSO on HepaRG differentiation, HEV-3 replication and IFN response. First, we investigated the gene and protein expression level of hepatocyte marker by RT-qPCR (albumin, HNF4α, G6PC) and by immunofluorescence (IF) microscopy (albumin). We confirmed an upregulation of the expression of hepatocyte-specific genes in HepaRG cells treated with DMSO at similar level than primary human hepatocytes. Interestingly, after DMSO removal, high levels of hepatocyte markers were maintained for up to 100 days, (albeit at a lower level than in the presence of DMSO). Despite the maintenance of a higher differentiation status, HEV viral load (RT-qPCR) and ORF2-specific staining (IF) were lower in the presence of DMSO (1 to 2-log). IFN response was investigated after HEV-3 infection in HepaRG. Upregulation of IFN or ISG gene expression was observed only late after infection (D100) and was more important in absence of DMSO. In conclusion, we have shown that HepaRG need to be cultured without DMSO for optimal HEV-3 replication contrasting with HBV for which the addition of DMSO is essential. Overall, this work provides a better characterization of the HepaRG cell line as a robust model to study HEV and its interaction with the innate immune system.