Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of early and late genes. This study finds that 0.31 mM protoapigenone from Thelypteris torresiana (Gaud.) inhibits the expression of EBV lytic proteins, including Rta, Zta, EA-D and VCA, in P3HR1 cells after lytic induction with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate. The lack of expression of EBV lytic proteins after protoapigenone treatment is attributed to the inhibition of the transactivation function of Zta because protoapigenone reduces the transactivation activity of Zta and Gal4-Zta, which contains the transactivation domain of Zta fused with Gal4. In contrast, protoapigenone does not affect the ability of Rta to activate a promoter that contains an Rta-response element, showing that the inhibition is unrelated to Rta. Furthermore, in a lactate dehydrogenase assay, protoapigenone is not toxic to P3HR1 cells at the concentrations that inhibit the function of Zta, showing that protoapigenone is valuable for studying the function of Zta and preventing EBV lytic proliferation.