Persistent infection of chicken anemia virus (CAV) in chickens has been suspected to result in immunosuppression and exogenous virus contamination within vaccine production. However, no direct evidence for persistent CAV infection has thus far been obtained. In this study, we aimed to establish an in vitro model of persistent CAV infection. CAV-infected MDCC-MSB1 (MSB1) cells, a Marek's disease virus-transformed continuous cell line, were cultured in the presence of both CAV and CAV neutralizing antibody (NA). Cell viability, expression of viral antigens, viral DNA, and recovery of CAV were examined by acridine orange/propidium iodide staining, immunofluorescence measurement, real-time PCR, and viral isolation, respectively. The results indicated that CAV was maintained and possibly replicated in CAV-infected cells cultured in the presence of NA, without affecting host cell viability. It was also shown that persistently infectious CAV induced cell death again after removing NA. The persistent infection of CAV in MSB1 cells was not related to viral gene mutation. In summary, we have herein established a novel model of persistent CAV infection in MSB1 cells cultured in the presence of NA.