Enterohemorrhagic E. coli (EHEC) O157:H7/H- (nonmotile) exist as Shiga toxin gene (stx)-positive and stx-negative variants in patients and the environment. We analyzed molecular characteristics, phenotypes and phylogenetic background of three stx-negative E. coli O157:H7/H- strains isolated from cattle and a pig and compared them with those of human EHEC and stx-negative E. coli O157:H7/H-. All three animal strains contained fliCH7 and two contained eae. One eae-positive strains (O157:H-) was sorbitol-fermenting (SF) and the other (O157:H7) was non-sorbitol-fermenting (nSF). These two strains shared a spectrum of non-stx putative virulence and fitness genes with human nSF and SF EHEC and stx-negative E. coli O157:H7/H- and belonged, similar to the vast majority of human isolates, to sequence type (ST) 11 in multilocus sequence typing. In contrast, the eae-negative O157:H7 animal isolate, which was SF, differed in spectrum of virulence genes and phylogenetically (ST717) from the two eae-positive strains and from human EHEC and stx-negative E. coli O157:H7/H-. In contrast to human stx-negative E. coli O157:H-, efforts to transduce the two stx-negative/eae-positive animal O157:H7/H- strains with stx2-encoding phages from human SF and nSF EHEC O157:H7/H- failed, despite the animal strains having intact loci where such phages integrate in human EHEC O157 (wrbA and yecE). The role of the animal stx-negative/eae-positive and stx-negative/eae-negative E. coli O157:H7/H- in their natural source and in human infections needs further investigation.