Uropathogenic Escherichia coli (UPEC) are one of the most common causes of urinary tract and bloodstream infections. UPEC possess key virulence determinants such as the capsule and lipopolysaccharide (LPS) that confer protection from host innate immune factors while advancing disease progression. Many of these virulence determinants are regulated by the transcriptional elongation factor RfaH, which binds to a specific motif known as the JUMPstart sequence located in the leader sequence of target virulence genes. RfaH binding enhances RNA polymerase activity and increases the efficiency of long operon transcription and translation. In this study, we investigated the RfaH regulatory network in UPEC using a combined bioinformatic and molecular (RNA-seq) approach. First, we built a HMM motif matrix using characterized JUMPstart sequences and applied this motif to predict JUMPstart sequences and hence, RfaH-regulated genes, in 1378 completely sequenced E. coli genomes. Next, the RfaH regulon was experimentally mapped in mid-log phase cultures of the UPEC ST131 strain S65EC using RNA-seq and validated with qRT-PCR. Overall, the matrix successfully identified all known JUMPstart sequences and detected a potentially new JUMPstart sequence belonging to an operon comprising 7 genes of unknown function. The RNA-seq analysis and qRT-PCR validation was congruent with the bioinformatic analysis, demonstrating that RfaH was required for the transcription of genes encoding the capsule, LPS and O-antigen. Overall, our study demonstrates that RfaH is a key regulator of multiple UPEC cell surface virulence determinants. Deciphering the function of RfaH will improve our understanding of UPEC pathogenesis and may enable the development of urgently needed anti-virulence therapeutics to address the enormous problem of increasing antibiotic resistance.