Escherichia coli ST131 is a globally disseminated, multidrug resistant uropathogenic E. coli (UPEC) clone responsible for a high proportion of urinary tract and bloodstream infections. Multiple factors including antibiotic resistance, superior gut colonization, serum resistance, enhanced virulence and high metabolic potential have been suggested to contribute to the global dominance of E. coli ST131 in community and hospital settings. In this study, we identified the molecular mechanisms required for growth of the E. coli ST131 reference strain EC958 in human urine (HU) using two complementary approaches: a genomics approach employing transposon-directed insertion-site sequencing (TraDIS) and a metabolomics approach. Using a hyper-saturated mini-Tn5 EC958 mutant library and TraDIS, we identified 24 genes (mapped to 20 metabolic pathways) required for growth in HU. We generated mutants with deletions in 14 of the genes and confirmed they were significantly attenuated for growth in HU compared to wild-type EC958. One of the genes required for growth in HU was crcB, which encodes a specific fluoride efflux pump. We measured the concentration of fluoride in HU as 1.2 mM, and confirmed the function of this gene using media supplemented with sodium fluoride. We also identified genes involved in the stringent response as required for growth in HU, and showed that a relA-spoT double mutant was significantly attenuated for colonization of the mouse bladder. Mutations in guaA, guaB and holC also led to reduced bladder colonisation in mice. Metabolomic analysis comparing the metabolites in HU before and after EC958 growth highlighted a significant reduction in L-lactate, indicating that L-lactate was used for growth. Mutations in glcA and lldP, the two known lactate permeases, showed that LldP was the major contributor to L-lactate uptake in EC958. Overall, our combined genomic and metabolomic analysis has provided new insights into UPEC growth in HU.