Objective To construct the adhesin gene iha mutant of uropathogenic Escherichia coli (UPEC) strain W140, and analyze the effects of iha deletion on UPEC biofilm formation. Methods Knockout of the iha gene was done with plasmid pKD46, pKD3, and pCP20 of Red recombinant system. pKD46 was transformed into W140 strain to express the three recombination proteins of λ phage. The chloramphenicol resistance gene flanked by flipase recognition target (FRT) of pKD3 was amplified by PCR and transformed into W140 strain to replace the iha gene. Finally pCP20 was transformed into W140 strain and expressed flipase recombinase, which deleted the chloramphenicol resistance gene between FRT sites. The differences of biofilm formation ability between the wild strain and the mutant were measured by crystal violet staining. Results PCR verification and DNA sequencing indicated that the iha gene was successfully knocked out and UPEC W140Δiha was constructed. The biofilm growth level of the mutant was significantly lower than that of wild strain (P<0.01). Conclusion The iha gene and its encoding product are involved in the process of UPEC biofilm formation. Suppressing iha gene may provide a new target for controlling urinary tract infection.