Abstract The Kaposi's sarcoma-associated herpesvirus (KSHV) genome replicates once per cell cycle, and the number of viral genome is maintained in the latency. The host cell-cycle-dependent replication of the viral genome is a fundamental process to critically keep the number of the genome. Here we show that the cellular pre-replication complex (pre-RC), the viral replication origin (ori-P) in a unit of the terminal repeat of the KSHV genome, and a viral replication factor, latency-associated nuclear antigen (LANA) accumulate at the nuclear matrix fraction in the G1 phase. We found not only that LANA itself was localized mainly to the nuclear matrix fraction but also that TR region of the KSHV genome existed together in the G1 phase. The localization of LANA at the nuclear matrix could be determined by structural consequence of the full length of LANA. Furthermore, transient replication assay revealed that the LANA's nuclear matrix localization was a pre-requisite for the efficient viral genome replication in the latency. Since LANA has been shown to bind the LANA binding sites (LBS) of the ori-P, these results suggest that LANA should recruit the ori-P to the nuclear matrix, where the complete pre-RC then forms on the ori-P, during the G1 phase. Thus, the nuclear matrix accumulation of cellular and viral replication factors is likely to be a key process for the initiation of replication of KSHV in the latency.