The objective of this longitudinal controlled trial was to determine the effect of systemic treatment with ceftiofur on antimicrobial susceptibility of fecal Escherichia coli isolates in dairy cows. Cows with metritis or interdigital necrobacillosis requiring systemic antimicrobial treatment were sequentially assigned to two treatment groups. The first group was treated with ceftiofur hydrochloride and the second with penicillin G procaine. Untreated healthy control cows were selected for sampling on the same schedule as treated cows. Fecal samples were collected on days 0, 2, 7, 14, 21, and 28. In total, 21983 E. coli isolates from 42 cows were analyzed for susceptibility to ampicillin, tetracycline, and ceftiofur using a hydrophobic grid membrane filter system to assess growth on agar containing selected antimicrobial drugs. Temporal changes in both the concentration of E. coli in feces and the susceptibility of E. coli to each drug were analyzed. A significant decrease in the concentration of fecal E. coli on days 2 and 7 post-treatment (but not thereafter) was detected in animals treated with ceftiofur. The proportion of all isolates (95% confidence interval in parentheses) showing reduced susceptibility at day 0 was 3.0% (2.5, 3.6) for ampicillin, 10.6% (9.7, 11.6) for tetracycline, and 4.8% (4.2, 5.6) for ceftiofur; 1.7% (1.3, 2.1) of isolates were resistant to ceftiofur based on growth at 8 μg/mL. Treatment did not have any significant effect on the proportion of isolates expressing reduced susceptibility to antibiotics with the exception of decreased tetracycline susceptibility in the ceftiofur-treated group on day 2. Although we found the potential for selection pressure by documenting the change in E. coli concentration after ceftiofur treatment, an increase in ceftiofur resistance was not found.