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The high cell density, immobilization and stability of biofilms are ideal characteristics for bacteria in resisting antibiotic therapy. CsgD is a transcription activating factor that regulates the synthesis of curly fimbriae and cellulose in <i>Escherichia coli</i>, thereby enhancing bacterial adhesion and promoting biofilm formation. To investigate the role of CsgD in biofilm formation and stress resistance in bacteria, the <i>csg</i>D deletion mutant Δ<i>csg</i>D was successfully constructed from the engineered strain <i>E. coli</i> BL21(DE3) using the CRISPR/Cas9 gene-editing system. The results demonstrated that the biofilm of Δ<i>csg</i>D decreased by 70.07% (<i>p</i> < 0.05). Additionally, the mobility and adhesion of Δ<i>csg</i>D were inhibited due to the decrease in curly fimbriae and extracellular polymeric substances. Furthermore, Δ<i>csg</i>D exhibited a significantly decreased resistance to acid, alkali and osmotic stress conditions (<i>p</i> < 0.05). RNA-Seq results revealed 491 differentially expressed genes between the parent strain and Δ<i>csg</i>D, with enrichment primarily observed in metabolism-related processes as well as cell membrane structure and catalytic activity categories. Moreover, CsgD influenced the expression of biofilm and stress response genes <i>pga</i>A, <i>mot</i>B, <i>fim</i>A, <i>fim</i>C, <i>ira</i>P, <i>omp</i>A, <i>osm</i>C, <i>suf</i>E and <i>ela</i>B, indicating that the CsgD participated in the resistance of <i>E. coli</i> by regulating the expression of biofilm and stress response. In brief, the transcription factor CsgD plays a key role in the stress resistance of <i>E. coli</i>, and is a potential target for treating and controlling biofilm.