Abstract: Objective To explore Cortex Phellodendri extract antibacterial activity against macrolides resistant mycoplasma pneumoniae (MP) in vitro, and to analyze its effect on the expression change of resistance gene MPN421 of MP. Methods The 23S rRNA mutations of 45 clinical isolated MP strains were determined by real-time PCR which were related to macrolides resistant. In further, MICs of 45 MP strains for Cortex Phellodendri extract were tested by serial dilution method. The MPNA421 mRNA expression change was measured with real-time PCR before and after being effected by Cortex Phellodendri extract. Results From 45 clinical isolated strains, there were 30 strains detected 23 s rRNA mutations, no mutations were detected in the other 15 strains. The MICs of 15 wild-type MP strains were 0.036-0.576 mg/ml for Cortex Phellodendri extract; the MICs of 30 resistant strains were 0.036-1.152 mg/ml. Combined with the MICs of berberine hydrochloride for most of the common bacteria and fungi were in range of 0.5-1.0 mg/ml. The results showed that 45 strains of MP for Cortex Phellodendri extract all were sensitive. On the condition without Cortex Phellodendri extract effect, the relative expressions of MPN421 mRNA of 15 wild-type MP strains and 30 MP resistant strains were 0.079 (0.071, 0.089), 0.083 (0.062, 0.084), P>0.05, respectively. After administration of Cortex Phellodendri extract for 6 hours or 9 hours, the relative expression of MPN421 mRNA of both wild-type strains and resistant strains were decreasing significantly (all P<0.01). Conclusion Both wild-type MP strains and macrolides-resistant strains were sensitive to Cortex Phellodendri extract. Cortex Phellodendri extract could decrease the relative expression of MPN421 mRNA, which mean that it could be used in treatment of the macrolides-resistant MP strain infection.