Human enterovirus 71 （EV71） is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease （HFMD） in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VPl-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TClD50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin （29.6 pmol/L） effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication： rheum emodin treatment decreased vira genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside （50 pmol/L） nor artemisinin （50 pmol/L） showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.