Epstein- Barr virus (EBV), a human herpesvirus, is several human lymphoid malignancies-associated. Our earlier study found the effect of Polygonum cuspidatum root on promoting EBV-positive apoptosis. Therefore, this study investigated the effects of the Polygonum cuspidatum ethyl acetate subfraction containing emodin on EBV gene expression and anti-EBV tumor cells. Resultantly, the the Polygonum cuspidatum ethyl acetate subfraction containing emodin ( F3a) promoted Raji cell death (50% cytotoxic concentration, CC50: 12.08 mu g/mL); the 12.5 mu g/mL F3a effect transcribed BRLF1 and BNLF1 and increased latent membrane protein 1 ( LMP1), which may reduce the intracellular phospho-extracellular signal-regulated kinase (ERK) and phospho-inhibitor of Nuclear factor kappa B a (I.Ba). Meanwhile, the Raji cells increased the intracellular reactive-oxygen species (ROS), activated the apoptosis-related proteins, cleaved caspase 3 and poly(ADP-ribose)polymerase (PARP), and increased the apoptosis percentage. Therefore, the Polygonum cuspidatum ethyl acetate subfraction containing emodin could be a therapeutic drug for EBV-related tumors.