Andrographis paniculata NEES is a medicinal plant that is commonly used in Asia. This work demonstrates that 25 microg/ml of ethanolic extract from A. paniculata (EEAP) and 5 microg/ml of andrographolide, a bioactive compound in EEAP, effectively inhibit the expression of Epstein-Barr virus (EBV) lytic proteins, Rta, Zta and EA-D, during the viral lytic cycle in P3HR1 cells. Transient transfection analysis revealed that the lack of expression of Rta, Zta and EA-D is caused by the inhibition of the transcription of BRLF1 and BZLF1, two EBV immediate-early genes that encode Rta and Zta, respectively. This study finds that the inhibition prevents the virus from producing mature viral particles. Meanwhile, andrographolide is not toxic to P3HR1 cells when the concentration is below 5 microg/ml, indicating that the compound is potentially useful as an anti-EBV drug.