Infected cells recognize viral replication as a DNA damage stress and elicit the ataxia telangiectasia-mutated (ATM)/p53-mediated DNA damage response signal transduction pathway as part of the host surveillance mechanisms, which ultimately induces the irreversible cell cycle arrest and apoptosis. Viruses have evolved a variety of mechanisms to counteract this host intracellular innate immunity. Kaposi's sarcoma-associated herpesvirus (KSHV) viral interferon regulatory factor 1 (vIRF1) interacts with the cellular p53 tumor suppressor through its central DNA binding domain, and this interaction inhibits transcriptional activation of p53. Here, we further demonstrate that KSHV vIRF1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. Detailed biochemical study showed that vIRF1 interacted with cellular ATM kinase through its carboxyl-terminal transactivation domain and that this interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a consequence, vIRF1 expression greatly reduced the level of serine 15 phosphorylation of p53, resulting in an increase of p53 ubiquitination and thereby a decrease of its protein stability. These results indicate that KSHV vIRF1 comprehensively compromises an ATM/p53-mediated DNA damage response checkpoint by targeting both upstream ATM kinase and downstream p53 tumor suppressor, which might circumvent host growth surveillance and facilitate viral replication in infected cells.