While a high osmolarity medium activates Cpx signaling and causes CpxR to represscsgDexpression, and efflux protein TolC protein plays an important role in biofilm formation inEscherichia coli,whether TolC also responds to an osmolarity change to regulate biofilm formation in extraintestinal pathogenicE. coli(ExPEC) remains unknown. In this study, we constructedΔtolCmutant and complement ExPEC strains to investigate the role of TolC in the retention of biofilm formation and curli production capability under different osmotic conditions. TheΔtolCmutant showed significantly decreased biofilm formation and lost the ability to produce curli fimbriae compared to its parent ExPEC strain PPECC42 when cultured in M9 medium or 1/2 M9 medium of increased osmolarity with NaCl or sucrose at 28°C. However, biofilm formation and curli production levels were restored to wild-type levels in theΔtolCmutant in 1/2 M9 medium. We propose for the first time that TolC protein is able to form biofilm even under high osmotic stress. Our findings reveal an interplay between the role of TolC in ExPEC biofilm formation and the osmolarity of the surrounding environment, thus providing guidance for the development of a treatment for ExPEC biofilm formation.