Abstract Hepatitis C virus (HCV) infection is a serious health problem worldwide that can lead to hepatocellular carcinoma or end-stage liver disease. Current treatment with pegylated interferon, ribavirin, and NS3/4A protease inhibitor would lead to a good prognosis in a large population of patients, but there is still no effective vaccine for HCV. HCV robustly infects hepatocytes in the liver. However, extrahepatic manifestations such as mixed cryoglobulinemia, a systemic immune complex-mediated disorder characterized by B-cell proliferation, which may evolve into overt B-cell non-Hodgkin's lymphoma, have been demonstrated. HCV-RNA is often found to be associated with peripheral blood lymphocytes, suggesting a possible interaction with peripheral blood mononuclear cells (PBMCs), especially B-cells with HCV. B-cell HCV infection was a matter of debate for a long time, and the new advance in HCV in vitro infectious systems suggest that exosome can transmit HCV genome to support "infection." We aimed to clarify the susceptibility of primary B-cells to HCV infection, and to study its functional effect. In this article, we found that the recombinant HCV J6JFH1 strain could infect human B-cells isolated from the peripheral blood of normal volunteers by the detection of both HCV-negative-strand RNA by reverse transcription polymerase chain reaction, and NS5A protein. We also show the blocking of HCV replication by type I interferon after B-cell HCV infection. Although HCV replication in B-lymphocytes showed lower efficiency, in comparison with hepatocyte line (Huh7) cells, our results clearly demonstrate that human B-lymphocytes without other non-B-cells can actually be infected with HCV, and that this interaction leads to the induction of B-cells' innate immune response, and change the response of these cells to apoptosis.