Raccoon dog parvovirus (RDPV) has spread widely in China, with gradually increasing morbidity and mortality. There is no commercial vaccine specifically for RDPV enteritis in China. Isolation of RDPV is very critical for the vaccine development against RDPV infection. The aim of this study was to isolate and identify RDPVs and analyze the phylogenetic clade of VP2 gene. The rectal swabs from raccoon dogs with severe diarrhea were screened by CPV colloidal gold strip, the positive swabs were treated and cultured in CRFK cells. The viruses in infected CRFK cells were identified by PCR detection, immunofluorescent test and electronic microscope observation. VP2 genes of the isolates were amplified and subjected to sequence. Two-month-old raccoon dogs were inoculated orally with the isolates for the virulence test. The results showed that one RDPV was isolated and named as RDPV HB01 strain. Sequence analysis revealed that the strain shared more than 99% nucleotide homologies with the canine parvovirus (CPV)-2a strains. All experimentally infected animals showed anorexia or depression with diarrhea in various degrees. Observation of histopathological lesions revealed that there were massive necrosis and abscission of villous epithelial cells in small intestine, loose and shedding lymphocyte structure in mesenteric lymph nodes, congestive medullary sinus and decreased lymphocyte of the white medullary lymph follicles in spleen. Therefore, the RDPV HB01 strain is classified as a CPV-2a variant and virulent to raccoon dogs.