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<i>Pasteurella multocida (Pm)</i> is one of the major pathogens of bovine respiratory disease (BRD), which can develop drug resistance to many of the commonly used antibiotics. Our earlier research group found that with clinical use of enrofloxacin, <i>Pm</i> was more likely to develop drug resistance to enrofloxacin. In order to better understand the resistance mechanism of <i>Pm</i> to enrofloxacin, we isolated <i>PmS</i> and <i>PmR</i> strains with the same PFGE typing in vitro, and artificially induced <i>PmR</i> to obtain the highly resistant phenotype, <i>PmHR</i>. Then transcriptome sequencing of clinically isolated sensitive strains, resistant and highly drug-resistant strains, treated with enrofloxacin at sub-inhibitory concentrations, were performed. The <i>satP</i> gene, of which the expression changed significantly with the increase in drug resistance, was screened. In order to further confirm the function of this gene, we constructed a <i>satP</i> deletion (<i>ΔPm</i>) strain using suicide vector plasmid pRE112, and constructed the <i>C-Pm</i> strain using pBBR1-MCS, and further analyzed the function of the <i>satP</i> gene. Through a continuously induced resistance test, it was found that the resistance rate of <i>ΔPm</i> was obviously lower than that of <i>Pm</i> in vitro. MDK₉₉, agar diffusion and mutation frequency experiments showed significantly lower tolerance of <i>ΔPm</i> than the wild-type strains. The pathogenicity of <i>ΔPm</i> and <i>Pm</i> was measured by an acute pathogenicity test in mice, and it was found that the pathogenicity of <i>ΔPm</i> was reduced by about 400 times. Therefore, this study found that the <i>satP</i> gene was related to the tolerance and pathogenicity of <i>Pm</i>, and may be used as a target of enrofloxacin synergistic effect.