Deficiency in biosynthesis of inner core of lipopolysaccharide (LPS) rendered a characteristic biofilm-forming phenotype inE.coli. The pathological implications of this new phenotype inShigella flexneri, a highly contagious enteric Gram-negative bacteria that is closely related toE.coli, were investigated in this study. The ΔrfaC(also referred aswaaC) mutant, with incomplete inner core of LPS due to deficiency in Hep biosynthesis, was characteristic of strong biofilm formation ability and exhibited much more pronounced adhesiveness and invasiveness to human epithelial cells than the parental strain and other LPS mutants, which also showed distinct pattern of F-actin recruitment. Failure to cause keratoconjunctivitis and colonize in the intestine in guinea pigs revealed that the fitness gain on host adhesion resulted from biofilm formation is not sufficient to offset the loss of fitness on survivability caused by LPS deletion. Our study suggests a clear positive relationship between increased surface hydrophobicity and adhesiveness ofShigella flexneri, which should be put into consideration of virulence ofShigella, especially when therapeutic strategy targeting the core oligosaccharide (OS) is considered an alternative to deal with bacterial antibiotics-resistance.