Little information on the epidemiology and pathogenicity of porcine enterovirus 9 (PEV-9) is available. The present study investigated the prevalence of PEV-9 in pig populations in middle and eastern China using reverse transcriptase (RT)-PCR. All 14 sampled farms were positive for PEV-9 and the overall prevalence of infection in the studied pigs was 8.3% (37/447). There was a higher frequency of infection in pigs aged 10-15 weeks (12/119, 10.1%) than in pigs aged >20 weeks (5/103, 4.9%). A 313 nucleotide sequence from the 5'-UTR region of 37 Chinese PEV-9 positive samples had 96.1-100% sequence homology. On phylogenetic analysis, sequences clustered into two major groups, from which two representative strains were selected to determine the complete RNA-dependent RNA polymerase (RdRp) gene sequence. Phylogenetic analysis based on the RdRp gene suggested that PEV-9 strains from China formed a new subgroup. Piglets were inoculated orally with the PEV-9 strain identified in this study. Although most experimental pigs showed no clinical signs, almost all carried PEV-9 in one or more tissues after 6 days post-inoculation. The results of tissue histologic examination suggested that PEV9 can cause pathological changes in cerebrum and lung.