The first proteomic characterization of the HepaRG cell line, the only cell line that is susceptible to hepatitis B virus (HBV) infection and supports a complete virus life cycle, is reported. Differential analysis of naive and HBV-infected HepaRG cells by two-dimensional gel electrophoresis revealed 19 differentially regulated features, 7 increasing and 12 decreasing with HBV infection. The proteins identified in these features were involved in various cellular pathways including apoptosis, DNA/RNA processing, and hepatocellular impairment. Similar expression changes in a number of the identified proteins have already been reported for other virus systems. Identification of these expression changes is a validation of the proteomics approach and contributes to an understanding of host cellular response to HBV infection.