Flagellin is a key component of the flagella of many pathogens, including Pseudomonas aeruginosa. Flagellin is an attractive vaccine candidate because it is readily produced and manipulated as a recombinant protein and has intrinsic adjuvant activity mediated through TLR5. Although DNA vaccines encoding native Pseudomonas B-type (FliC) or A-type (FlaA) flagellin are strongly immunogenic, the resultant Ab response interferes with the interaction of homologous flagellin with TLR5. This reduces the ability of the host to clear homologous, but not heterologous, flagellin-expressing P. aeruginosa. To circumvent this problem, a DNA vaccine encoding a mutant FliC R90A flagellin was developed. The mutant Ag encoded by this vaccine was highly immunogenic, but its ability to interact with TLR5 was reduced by >100-fold. Vaccination with this flagellin mutant DNA vaccine induced cross-reactive Abs against both FliC and FlaA, but few Abs capable of interfering with TLR5 activation. The flagellin mutant DNA vaccine provided excellent protection against both FliC- and FlaA-expressing P. aeruginosa. These findings suggest that vaccines against flagellated pathogens should avoid inducing Abs against TLR5 and raise the possibility that flagellated bacteria evade host elimination by facilitating the production of Abs that reduce the host's ability to mount an innate immune response.