Objective:To establish the model of human herpesvirus (HHV)-6A subtype latent infection of human glioma cell line U251 in vitro. Methods: U251 cells infected by HHV-6A were cultured and passaged for model establishment. The cell morphology changing, virus replication and viral gene expression were detected to identify the latent infection stage. Results: With increase of HHV-6A infection of U251 cells algebra, the viral gene copy number was gradually decreased to a stable low level. The HHV-6A U94 gene, a representative of the latent stage of infection of the virus, was gradually decreased and then maintained at a stable level. Conclusion: HHV-6A was successfully transferred from the acute infection stage to the latent infection stage in U251 cells in vitro.