Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.