The effects of different concentrations of 5-fluorouracil, rifampicin and tetracycline on the phytoplasmas in the Paulownia tomentosa×Paulownia fortunei seedling and the pathogenic protein related to the witches’ broom symptom were studied with PCR based on 16S rRNA gene of the phytoplasmas and 2-dimensional electrophoresis of proteins. The results indicated that the plantlets in vitro culture treated with 0.5~300 mg·L -1 5-fluorouracil had little effect on the symptom remission, but those plantlets treated with 100 and 150 mg·L -1 rifampicin for 30 d showed obvious effect on the symptom remission respectively, and the phytoplasmas in the plantlets treated with 150 mg·L -1 rifampicin could not be detected with PCR of 16S rDNA of the phytoplasmas. Moreover, the protein (pI6.8,m 24 ku) which appeared in both the healthy seedling and diseases ones treated with 150 mg·L -1 rifampicin disappeared in the infected seedling and the those without the symptom after treating with rifampicin and tetracycline, and in which existed with phytoplasma through PCR detection.