Extracellular vesicles (EVs) are involved in cell-to-cell communications involving a molecular cargo comprising enzymes, toxins, immune decoys, nutrients, genetic material and/or signals that influence gene expression. EV communications can be intraspecies, interspecies and interkingdom, but few examples of EV-mediated communication between human cells and bacteria exist. We have investigated the effects of EVs from the human colorectal adenocarcinoma cell line HT29 on pathogenic (diarrhoeagenic) and probiotic strains of Escherichia coli. HT29-EVs were isolated from conditioned media collected from a CELLine bioreactor by differential ultracentrifugation and purified by size exclusion chromatography. EVs were verified by TEM and western blot to show the presence of CD81, TSG101 and the absence of GRP94. A dose of HT29 EVs that did not affect growth was used to challenge pathogenic and probiotic strains of E. coli in early log-phase and RNA was isolated after 2 h incubation with EVs in iron-replete and iron-restricted conditions, with 5 replicate cultures for each combination. RNAseq transcriptomic analysis was applied to identify bacterial genes influenced by the EV-challenge. The bacterial responses reflected in the transcriptome did not indicate widespread changes, or many differences between the pathogenic and the probiotic. The changes that were observed (>2-fold change; p<0.05) identify roles for molybdenum (Mo) uptake and metabolism, hydrogenase-2 activity and a variety of oxidative stress responses.