The globally disseminated multidrug-resistant uropathogenic E. coli ST131 clone is a major cause of urinary tract infection and life-threatening sepsis. To unravel the adaptive features that have led to its overriding dominance, we conducted a comprehensive transcriptome analysis of the representative ST131 strain EC958 under various infection-relevant conditions. Using RNA-seq, we examined transcriptomic responses of EC958 to two growth phases (mid-exponential and early stationary), five physiological shocks (high salt, low pH, low iron, anaerobic and human urine), and three antibiotic shocks (ciprofloxacin, cefotaxime and fosfomycin). Our analysis revealed that 68% of the genome (3,463 genes) were expressed across all conditions, with distinct expression signatures for each physiological condition and a common response to antibiotics. Differential RNA-seq mapping of transcription start sites led to the discovery of more than 200 novel noncoding RNA (ncRNA) candidates. Several were confirmed by Northern blotting, with their abundance correlating with RNA-seq data. Notably, the transcriptomic responses to ciprofloxacin and low iron conditions were similar. This prompted further investigation using CLASH (UV cross-linking, ligation, and sequencing of hybrids), which identified 780 ncRNA-mRNA interactions, including 57 involving differentially expressed ncRNA or mRNA. Functional studies of two ncRNAs yielded significant findings. Mutation of AS_pc02 increased EC958 resistance to colicins Ia and Ib, likely due to downregulation of the colicin receptor gene cirA. Mutation of a second ncRNA, srna1313, which targets an O-antigen gene encoding a glycosyl transferase, led to reduced colonisation of EC958 in a mouse UTI model. This study provides a comprehensive transcription signature of ST131, offering crucial insights into its adaptability to infection-relevant stresses and highlighting the potential role of ncRNAs in ST131 pathogenesis.