E. coli O157:H7 colonizes the bovine intestine, can contaminate food through fecal shedding, and causes human diarrheal and systemic illnesses. Catabolism of particular carbohydrates by E. coli has been found to be important for intestinal colonization of mice. In this study, we assessed whether catabolism of two mucin-derived carbohydrates are important for E. coli O157:H7 colonization of adult cattle. This was accomplished by competitively co-colonizing streptomycin-treated adult cattle with a wild-type strain of E. coli O157:H7 and isogenic mutants in catabolic pathways for mucin-derived carbohydrates N-acetylgalactosamine or l-fucose. Both mutants colonized poorly compared to the wild-type during the initiation stage of colonization (days 0-6). During the maintenance stage of colonization (days 7-15), the mutant unable to use N-acetylgalactosamine did not show a colonization defect, whereas the strain unable to use fucose had a significant colonization defect. These results support the concept that growth and colonization of E. coli O157:H7 in the bovine rectum has a nutritional basis, with a nutrient preference for l-fucose over N-acetylgalactosamine.