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AIM: To investigation the anti-coxsackievirus B3 (CVB3m)effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB3m.METHODS: HeLa cells were infected with CVB3m and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A600 was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A600.RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration.Consistent results were got through these two methods.We also investigated the mechanism of its anti-CVB3m effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB3m attachment, entry, biosynthesis and assemble in cells.CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB3m in a dose-dependent manner and this effect exists through the process of CVB3m attachment, entry, biosynthesis and assemble in cells,suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.