To assess the potential for food contamination with Clostridium difficile from food animals, we conducted a cross-sectional fecal prevalence study in 944 randomly selected cattle harvested at seven commercial meat processing plants, representing four distant regions (median distance of 1,500 km) of the United States. In all, 944 animals were sampled in the summer of 2008. C. difficile was isolated from 1.8% (17 of 944) of cattle, with median fecal shedding concentration of 2.2 log CFU/g (range = 1.6 to 4.8, 95% confidence interval = 1.6, 4.3). Toxigenic C. difficile isolates were recovered from only four (0.4%) cattle. One of these isolates was emerging PCR ribotype 078/toxinotype V. The remaining toxigenic isolates were toxinotype 0, one of which was an isolate with resistance to linezolid, clindamycin, and moxifloxacin (by the E-test). All isolates were susceptible to vancomycin, metronidazole, and tigecycline, but the MICs against linezolid were as high as the highest reported values for human-derived isolates. The source of the linezolid-clindamycin-moxifloxacin resistance in a toxigenic C. difficile isolate from cattle is uncertain. However, since the use of these three antimicrobial agents in cattle is not allowed in North America, it is possible that resistance originated from an environmental source, from other species where those antimicrobial agents are used, or transferred from other intestinal bacteria. This study confirms that commercial cattle can carry epidemiologically relevant C. difficile strains at the time of harvest, but the prevalence at the time they enter the food chain is low.