A survey was conducted on lxodes sinensis and its parasitism hosts for the infection of Borrelia burgdorferi with PCR and cultivation method. Only one spirochete strain was isolated from 40 I. sinensis collected in Huangshan Mountains. While all blood sample and other I. sinensis ticks fail to get Lyme spirochetes isolation. When detected by 16s rRNA amplification, 6.78% female and 5% male adult I. sinensis ticks yield positive result, two of 92 Chinese hares and one of 16 Chinese muntjaes also appear to be positive with the same DNA band, while all nymph ticks appeared uninfected. However, these DNA amplicon have the same 16S rRNA sequences without any difference. The isolated spirochetes strain obtained from I. sinensis in Huangshan show high similarity to other strains of B. afzelii by phylogeny analysis with 16S rRNA sequences and might belong to Borrelia afzelii species.The finding together with the proved transmission ability, imply that I. sinensis might be the vector tick species in southern China.