Murine gammaherpesvirus-68 (MHV-68), a close homologue of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can infect many cell lines efficiently and is able to infect laboratory mice, and has emerged as an excellent model for studying its human counterparts. The replication and transcriptional activator (RTA), mainly encoded by open read frame 50 (ORF50), is a conserved immediate-early protein in gammaherpesviruses. RTA initiates the viral lytic cycle by activating the expression of downstream viral lytic genes. Therefore，it is important to investigate the mechanism by which RTA regulates downstream gene expression. Two RTA dependent promoters of MHV-68 had previously been reported, however, the mechanism of the interaction between RTA and the promoters was not characterized. Our group has previously identified a new RTA responsive element (named RRE-B) in MHV-68 and characterized in detail the mechanisms involved. In the study, we first analyzed the MHV-68 genome and identified a sequence homologous to RRE-B in the promoter region of OFR9, and designated it as ORF9p-RRE. Dual luciferase reporter assay showed that RTA could activate the ORF9 promoter and this activation was dependent on ORF9p-RRE. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) further demonstrated that RTA bound to ORF9p-RRE in vitro and in vivo. In summary, we showed here that the promoter of MHV-68 ORF9 is responsive to RTA activation through ORF9p-RRE. The study provides support to further investigate and understand the role of RTA in regulating viral gene expression and viral life cycle.