Sophocarpine (SCA) is a bioactive alkaloid present in Sophoraflavescens Ait. The present study demonstrated that SCA inhibited enterovirus 71 (EV71) infection in Vero cells. The results indicated that the 50% cytotoxicity concentration of SCA for Vero cells was 1,346 µg/ml, and the 50% inhibition concentration of SCA against EV71 was 350 µg/ml. SCA produced a marked inhibitory effect against EV71 when the Vero cells were treated with SCA prior to infection with the virus. Additionally, SCA was effective against EV71 when the Vero cells were infected with EV71 (100xTCID50) that had been treated with SCA for 2 h, and was effective when the Vero cells were infected with EV71 (100xTCID50) at 37°C under 5% CO2 for 2 h prior to treatment with SCA for 2 h. SCA was demonstrated to inhibit the attachment and penetration of EV71 and was more effective at inhibiting attachment. The assay additionally verified that SCA suppressed the replication of viral genomic RNA and indicated that SCA may inhibit EV71 infection in vitro.