Avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC) harbour common virulence factors in spite of being associated with disease in different hosts. APEC strains have been shown to have zoonotic potential. In contrast, it is not known whether UPEC strains can cause infection in immunologically competent hens. The objective of the current study was to compare the ability of the well-characterized UPEC strain, UTI89, and the APEC strain, F149H1S2, to infect human and avian cells in culture and to cause salpingitis in an infection model in adult laying hens. In vitro characterization showed that the strains grew equally well in human urine, and both were able to infect human intestinal (Int407) and bladder (J82) epithelial cell lines, and they survived in avian macrophages (HD11) to the same extent. Groups of adult birds were inoculated with 10⁸ bacteria directly into the oviduct using a surgical procedure. After an infection period of 48 h, bacterial load in the oviduct was determined by dilution series, and pathology was determined based on gross lesions and histological observations. Similar counts of UPEC UTI89 (ST95) and the APEC strain F149H1S2 (ST117) were obtained from tissues of infected birds, and salpingitis as evaluated by clinical score and histopathology was observed to a similar extent after infection with the two strains. Together, the results showed that UPEC UTI89 and APEC F149H1S2 have a similar potential for causing salpingitis in laying hens in the model used. No infection differences were observed between the UPEC UTI89 wild type and a mutant strain with knock-out of the well-known virulence gene, fimH, (UPEC UTI89ΔfimH), showing that the salpingitis model is not suitable for the detection of all UPEC virulence factors.