Escherichia coli (E. coli) is perhaps the most studied of all model organisms. Pathogenic strains, such as E. coli O157:H7, pose a significant risk to human health. Recent findings using whole-genome expression profiling revealed what E. coli MG1655 genes were induced by the nutrients available in the mammalian intestine. The mutational analysis/microarray study found that only mutations in sugar pathways affected the colonization of the bacteria. The goal of this study was to determine the in vitro metabolism preference of two different colonizing E. coli strains (MG1655 and EDL933) towards the 14 monosaccharides known to be found in intestinal mucus. Carbohydrate concentrations were monitored using capillary electrophoresis (CE) with different detection methodologies. The disappearance of each sugar in the growth media provided the nutritional preference of E. coli. No single capillary electrophoresis method could be developed to monitor all the carbohydrates simultaneously. Instead, three independent CE methods were required. The combination of CE-UV and CE-MS data provided a complete picture of the actual carbohydrate metabolism of E. coli from colonization to constant phase. The data showed that different strains have different nutritional preferences. MG1655 did not metabolize N-actylgalactoseamine, while EDL933 did. Other sugars changed in their order of metabolism. The in vitro model clearly indicates that certain sugars are metabolized only during colonization and others during maintenance. This in vitro model provided an enhanced view of the actual carbohydrate preference of two different strains of E. coli and demonstrated significant differences between the commensal and pathogenic strains.