Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in animals and humans. We applied Transposon-Directed Insertion Site sequencing (TraDIS) to determine the fitness genes in two well-characterized UPEC strains, UTI89 and CFT073, in order to identify fitness factors during UTI in a pig model. This novel animal model better reflects the course of UTI in humans than the commonly used mouse model, and facilitates the differentiation between sessile and planktonic UPEC populations. A total of 854 and 483 genes in UTI89 and CFT073, respectively, were predicted to contribute to growth in pig urine, and 1257 and 764, were scored as required for colonization of the bladder. The combined list of fitness genes for growth in urine and cystitis contained 741 (UTI89) and 439 (CFT073) genes. The essential genes for growth on LB agar media supplemented with kanamycin and the fitness factors during growth in human urine were also analyzed in CFT073. A total of 457 essential genes were identified and the pool of fitness genes for growth in human urine included 215 genes. The gene rfaG, which is involved in lipopolysaccharide biosynthesis, was included in all the fitness-gene-lists and was further confirmed to be relevant for all the conditions tested regardless of the host and the strain. Thus, this gene may represent a promising target for the development of new therapeutic strategies against UTI UPEC-associated. Besides this important observation, the study revealed strain-specific differences in gene-essentiality as well as in the fitness-gene-repertoire for growth in human urine and UTI of the pig model, and it identified novel factors required for UPEC-induced UTIs.