Polygonum cuspidatum is widely used as a medicinal herb in Asia. In this study, we examined the ethanolic extract of Polygonum cuspidatum roots (PcE) for their capacity to inhibit the EBV lytic cycle. The cell viability was determined by the MTT [3-(4,5- dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The expression of Epstein-Barr virus (EBV) lytic proteins was analyzed by flow cytometric assay. Real-time quantitative PCR was used to measure the EBV virion production. Transient transfection analysis was subsequently performed to assess the transcription of lytic genes, including BRLF1 and BZLF1. Results showed that the PcE inhibited the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduced the production of virus particles, showing that PcE is useful for preventing the proliferation of the virus.