Biological processes originating from the DNA and RNA can be regulated by the secondary structures present in the stretch of nucleic acids, and the G-quadruplexes are shown to regulate transcription, translation, and replication. In this study, we identified the presence of multiple G-quadruplex sites in the region (oriLyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) DNA, which is essential for DNA replication during the lytic cycle. We demonstrated the roles of these G-quadruplexes through multiple biochemical and biophysical assays in controlling replication and efficient virus production. We demonstrated that KSHV achieves this by recruiting RecQ1 (helicase) at those G-quadruplex sites for efficient viral DNA replication. Analysis of the replicated DNA through nucleoside labeling and immunostaining showed a reduced initiation of DNA replication in cells with a pharmacologic stabilizer of G-quadruplexes. Overall, this study confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be exploited for controlling viral DNA replication.