Uropathogenic Escherichia coli (UPEC) is the primary causative agent of uncomplicated urinary tract infections (UTIs). UPEC fitness and virulence determinants have been evaluated in a variety of laboratory settings that include a well-established mouse model of UTI. However, the extent to which bacterial physiology differs between experimental models and human infections remains largely understudied. To address this important question, we compared the transcriptomes of three different UPEC isolates in human infection and a variety of laboratory conditions including LB culture, filter-sterilized urine culture, and the UTI mouse model. We observed high correlation in gene expression between the mouse model and human infection in all three strains examined (Pearson correlation coefficient of 0.86-0.87). Only 175 of 3,266 (5.4%) genes shared by all three strains had significantly different expression levels, with the majority of them (145 genes) down-regulated in patients. Importantly, gene expression of both canonical virulence factors and metabolic machinery were highly similar between the mouse model and human infection, while the in vitro conditions displayed more substantial differences. Interestingly, comparison of gene expression between the mouse model and human infection hint at differences in bladder oxygenation as well as nutrient composition. In summary, our work strongly validates the continued use of this mouse model for the study of the pathogenesis of human UTI.