Sequence type (ST)73, ST95, and ST131 belonging to phylogroup B2 of E. coli are the most prevalent lineages isolated from humans worldwide, causing most extra-intestinal infections in humans and other vertebrates [1,2]. Survey data shows that at the host population level that these three STs can co-exist, yet the data also indicates that these STs do not co-occur in the same host. To understand the ecological niches of these STs, this study investigated the phenotypic characteristics 48 isolates from humans of each ST. Substrate utilisation, heat stress tolerance, adhesion assay, and bacteriocin production experiments were performed. We found that 69% ST131 strains could exploit all substrates, 40% ST95 strains could not utilise any substrates, while the ability of ST73 strains varied and depended on substrates. ST73 strains were the most resistant at 47oC (65%). However, only 31% of ST95 strains and 33% of ST131 strains were tolerant at 47oC. Furthermore, 64% strains from ST73 could invade into Caco-2 cells compared to ST95 (25%) and ST131 (36%). In bacteriocin tests, after testing with sensitive strains, 22 ST73, 28 ST95, and 12 ST131 strains produced bacteriocins. Then, these 62 producers were tested with 144 indicator strains from the three STs. The results indicated that ST73 strains were the strongest “killers”. Moreover, 85% ST73 strains were resistant to more than 80% producers, followed by 75% of ST95 and 73% of ST131 strains. In summary, ST131 strains could utilise almost all substrates. ST73 strains are tolerant to heat stress, but also could invade into Caco-2 cells, and inhibit the growths of strains from ST95 and ST131 by producing bacteriocins. By contrast, ST95 strains had the lowest capacity to utilise substrates, to invade into host cells, and to resist to heat stress. These differences contribute to the distinct ecological niches of strains from ST73, ST95, and ST131.