Objective To test a novel baculovirus expressing Epstein-Barr virus (EBV) lytic replication immediate early gene Rta, in combination with antiviral chemical ganciclovir, for the activation of latent EBV in nasopharyngeal carcinoma (NPC) cells, and the killing of tumor cells. Methods A baculovirus vector with EBV replication origin OriP, and the Rta expression cassette led by CMV promoter, was used to treat EBV-latently infected NPC cell line Hone1-EBV, as well as nude mice tumor model established from the cell line, in combination with ganciclovir. The effect on the growth of cell and tumor was studied. Results When treated with recombinant baculovirus in combinantion with ganciclovir, the growth of EBV-positive cells was significantly reduced to only 51% of the control. In the nude mice experiments, tumor growth was also greatly reduced. At 10th day after the first injection, the average volume and weight of the treated group was only about 20% and 30% of that of the control group, respectively. Histological analysis indicated there was significant cell necrosis in the baculovirus-ganciclovir-treated tumor tissue. Conclusion Co-application of RTA-expressing baculovirus vector and gancyclovir may be useful for the therapy of EBV associated NPC.