To investigate the antiviral activity and potential mechanism of carboxymethylpachyman(CMP) against enterovirus 71(EV71), the effect of CMP on the proliferation of RD cells was detected by CCK-8 method. The cytopathic effect(CPE) method was used to observe the inhibitory effect of CMP on the CPE caused by EV71 infection. The median tissue culture infective dose(TCID50) method was used to detect the inhibitory level of CMP on EV71 infectious virus particles. The expression of EV71 VP1 protein was detected by RT-qPCR. The expressions of VP1, P62, LC3, Caspase-3 and cleaved Caspase-3 protein were analyzed by Western blotting. The results showed that different mass concentrations(0, 250, 500, 1 000 μg/mL) of CMP had no significant cytotoxicity on RD cells. Compared with the control group, CMP effectively inhibited the replication of EV71 in the range of non-toxic concentrations of EV71 replication and dose-dependently reduced the cytopathic effect caused by viral infection. CMP dose-dependently reduced the expression of EV71 viral structural protein VP1 and decreased the production of infectious viral particles in the offspring. CMP down-regulated the expression of LC3-II and cleaved Caspase-3 after EV71 virus infection of RD cells.