The pandemic, multidrug resistant clonal lineage of Escherichia coli, known as ST131, is a major uropathogen associated with urinary tract and bloodstream infections both in hospitals and the community. Type 1 fimbriae are the major surface-expressed virulence factor in uropathogenic E. coli (UPEC) important for initiating bladder infection. Previous genomic and molecular analyses of clinical ST131 isolates from around the world indicated that regulation of type 1 fimbriae might be unique to this pandemic lineage. Using a novel read-mapping approach based on Illumina sequencing, we have confirmed that its regulation and expression in the clinically predominant sub-lineage of ST131 (clade C/H30-R) is indeed significantly different to non-ST131 UPEC and other less prevalent ST131 groups. This is interesting given that we also found type 1 fimbriae to be a critical determinant for ST131 bladder colonisation and biofilm formation. The ability of different ST131 strains to form biofilms varied within our isolate collection and was greatly influenced by different growth conditions. However, inhibition of type 1 fimbriae-mediated adhesion by a chemical blocker completely eliminated biofilm formation. A genetic screen further revealed that the expression of type 1 fimbriae was closely linked to the metabolic state of the bacteria. Thus, understanding the factors contributing to the widespread pathogenic success of ST131 is critical to the development of effective intervention strategies that can reduce its current global burden.