Plants are increasingly considered as secondary reservoirs for commensal and pathogenic Escherichia coli strains, but the ecological and functional factors involved in this association are not clear. To address this question, we undertook a comparative approach combining phenotypic and phylogenetic analyses of E. coli isolates from crops and mammalian hosts. Phenotypic profiling revealed significant differences according to the source of isolation. Notably, isolates from plants displayed higher biofilm and extracellular matrix production and higher frequency of utilization of sucrose and the aromatic compound p-hydroxyphenylacetic acid. However, when compared with mammalian-associated strains, they reached lower growth yields on many C-sources commonly used by E. coli. Strikingly, we observed a strong association between phenotypes and E. coli phylogenetic groups. Strains belonging to phylogroup B1 were more likely to harbour traits indicative of a higher ability to colonize plants, whereas phylogroup A and B2 isolates displayed phenotypes linked to an animal-associated lifestyle. This work provides clear indications that E. coli phylogroups are specifically affected by niche-specific selective pressures, and provides an explanation on why E. coli population structures vary in natural environments, implying that different lineages in E. coli have substantially different transmission ecology.