Chimeric-antigen-receptors (CARs) are synthetic receptors designed to drive antigen-specific activation of T cells upon binding to cognate antigen. CAR-T cells are used in cancer therapy but are also interesting for chronic viral infections. Our study aimed to generate novel CARs that target the hepatitis B virus envelope protein (HBVenv) using an antigen-binding fragment (Fab) consisting of heavy and light chains instead of a variable single-chain fragment. The aim of this study was to overcome functional alterations of the new format and study the antiviral efficacy of FabCAR-engrafted T cells in vitro and in vivo.We constructed novel CARs containing the Fab fragment of HBVenv-specific monoclonal antibodies as binding domains and CD3 as well as CD28 intracellular signaling domains. Multifunctional FabCAR-T cells could be induced via HBsAg stimulation. FabCAR-T cells specifically eliminated HBVenv transgenic cell lines Huh7S and HepG2SML. FabCAR-T cells showed antiviral activity by significantly decreasing the level of viral antigen, intracellular HBV DNA, and HBV cccDNA in HBV-infected HepG2-NTCP cells in vitro. To study in vivo efficacy, CD45.1 murine T cells expressing FabCAR were transferred to CD45.2 AAV-HBV infected, HBV-carrier Rag1 knock-out mice; after adoptive transfer, FabCAR-T cells proliferated and localized to the liver, resulting in target cell killing indicated by ALT flare and an antiviral effect by HBsAg and HBeAg reduction.T cells stably transduced with our FabCARs are polyfunctional and have antiviral effects in cell culture and in a preclinical animal model, which are promising candidates for treating chronic hepatitis B and HBV-associated hepatocellular carcinoma.