Proteus mirabilis is a facultative Gram-negative uropathogen. The nitrogen-metabolic phosphotransferase system, PTSNtr, consists of the enzymes INtr, NPr and IIANtr that are encoded by ptsP, ptsO, and ptsN, respectively. EIIANtr has been found to be related to the regulation of potassium and phosphate uptake, and its phosphorylation may have different regulatory effects. In this study, we suggested that P. mirabilis tends to be regulated by phosphorylated- EIIANtr during swarming, and it’s also inhibit the ability of H2O2, acid and salt resistance. Next, the reporter assay indicate that phosphorylated- EIIANtr inhibits the signal transduction of PhoRB in a low-phosphorus environment. Finally, we found that the ptsN mutant and ptsN (H72A) were increase the ability of H2O2, acid and salt resistance in a low-phosphorus environment. In conclusion, we found that the EIIANtr and its phosphorylation regulates the motility and the ability to resisit H2O2, acid and salt of P. mirabilis. Furthermore, the two-component system PhoRB involve in modulating the tolerance to H2O2, acid and high salt with Phosphorylated- EIIANtr. This is the first study to investigate the impact of EIIANtr and its phosphorylation status on the pathogenicity of P. mirabilis.