This study aims to explore the in vitro and in vitro synergistic effect of palmatine (PA) combined with cefoperazone (CPZ) against extended spectrum β-lactamases (ESBLs) producing Escherichia coli, and to explore the possible mechanisms of the two drug combinations through pharmacodynamics and pharmacokinetics. Screening of multidrug-resistant ESBLs-producing E. coli by paper diffusion (K-B) and polymerase chain reaction and determination of genotypes. Quantification of the synergistic antimicrobial strength of PA in vitro by K-B and checkerboard methods. Molecular docking, cefonidothiophene hydrolysis assay, and three-phase hydrolysis for analysis of the effect of PA on the enzyme activity. Construction of a rat bacterial infectious pneumonia model to examine the interplay between the in vivo protective effect of PA combined with CPZ and pharmacokinetics. A CTX-M multidrug-resistant ESBLs-producing strain of E. coli 2022-72 was screened in the experiment; the minimal inhibit concentration (MIC) of CPZ was reduced from 32 μg/mL to 8 μg/mL, and the MIC of PA was reduced from 256 μg/mLto 32 μg/mL after the combination.The fractional inhibitory concentration was 0.375, indicating the synergistic effect of the combination of CPZ and PA; Molecular docking results showed that PA had good binding activity with CTX-M-type ESBLs, with a binding energy of -7.2 kcal/mol, cefonidothiophene hydrolysis test, and three-phase hydrolysis test both showed that PA exhibited antimicrobial coordination synergism by inhibiting ESBLs activity.The combination of the two drugs significantly improved the levels of inflammatory factors in the serum of animals with bacterial infection pneumonia model, including a significant decrease in C-reactive protein (P<0.05), interleukin-6, tumour necrosis factor-α had a highly significant decrease (P<0.01), and the effect on lung bacterial inhibition was significantly better than that of the drugs alone (P<0.01), and the strongest inhibitory effect on histopathological changes in the lungs was observed. The results of pharmacokinetic experiments show that the combination did not alter the pharmacokinetic parameters of the individual drugs.In conclusion, PA can enhance the exogenous and ex vivo antimicrobial effects of CPZ by decreasing the activity of ESBLs and effectively increase the in vivo anti-infective protection, and the synergistic effect has no significant correlation with pharmacokinetics.