Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, they undergo metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. To understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of the UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, most prominently arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were likewise upregulated together with proteins involved in sulfur compound turnover. Ribosomal proteins were downregulated relative to growth in MOPS in this environment. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.