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Avian pathogenic <i>Escherichia coli</i> (APEC) is one of the most common pathogens in poultry and a potential gene source of human extraintestinal pathogenic <i>E. coli</i> (ExPEC), leading to serious economic losses in the poultry industry and public health concerns. Exploring the pathogenic mechanisms underpinning APEC and the identification of new targets for disease prevention and treatment are warranted. <i>YgeK</i> is a transcriptional regulator in APEC and is localized to the type III secretion system 2 of <i>E. coli.</i> In our previous work, the transcription factor <i>ygeK</i> significantly affected APEC flagella formation, bacterial motility, serum sensitivity, adhesion, and virulence. To further explore <i>ygeK</i> functions, we evaluated its influence on APEC biofilm formation and resistance to environmental stress. Our results showed that <i>ygeK</i> inactivation decreased biofilm formation and reduced bacterial resistance to environmental stresses, including acid and oxidative stress. In addition, the multi-level regulation of <i>ygeK</i> in APEC was analyzed using proteomics, and associations between differentially expressed proteins and the key targets of <i>ygeK</i> were investigated. Overall, we identified <i>ygeK</i>’s new function in APEC. These have led us to better understand the transcriptional regulatory <i>ygeK</i> and provide new clues about the pathogenicity of APEC.