Cattle are the main reservoir for E. coli O157 and the majority of food-borne illnesses associated with this organism are attributed to the consumption of foods of bovine origin. To date, little work has been undertaken to understand the physiological state of E. coli in complex food matrices such as meat. Published reports also suggest that there are regional variations in E. coli O157 genotypes and this may provide a basis for the different disease burdens observed globally. It is therefore reasonable to predict that the different genotypes possess genetic repertoires that respond uniquely to the environmental conditions experienced in the meat production environment. This study analysed the transcriptome of E. coli on beef during attachment at a low temperature relevant to conditions experienced during carcass chilling. The aim of the study was to identify genes that may be associated with growth and survival on the beef carcass and, to compare the response of an E. coli isolate representative of the general genetic ‘type’ found in Australia to that of a pathogenic clinical isolate not typically found in Australia. The RNA-Seq study revealed a common gene expression response elicited by both strains, with the most highly up-regulated genes including those involved in the response to acid, osmotic, oxidative and cold stress. Both strains also up-regulated genes involved in multidrug resistance and encoding Shiga toxin subunits. The E. coli isolates also displayed unique gene expression profiles on meat, indicating that the genotypes may differ in their ability to persist in the meat production environment and therefore also in their ability to cause disease.