In order to determine the principal vectors of Lyme disease in South China, the capability of Ixodes sinensis to transmit Lyme disease spirochetes transstadially was estimated in the laboratory. Results suggest that I. sinensis can acquire active Lyme disease spirochetes by feeding on infected mice of Kunming strains (KM), and the positive rates of larvae and nymph I. sinensis infected were 94.0% and 92.0%, respectively. I. sinensis maintained active spirochetes during blood digestion and molting periods; the subsequent questing tick stages were also found to be infected and infectious to naive KM mice, which resulted in more than 66.7% spirochete-positive KM mice. I. sinensis can transmit the Lyme disease spirochete Borrelia garinii from infected KM mice to naive KM mice during larvae-nymph and nymph-adult periods. Therefore, I. sinensis might be considered as a competent vector of Lyme disease in South China.