Objective - To determine effects of administration of ceftiofur crystalline-free acid (CCFA) on antimicrobial susceptibility of Escherichia coli in feedlot cattle. Animals - 61 feedlot steers. Procedures - A cohort study was conducted. Steers were housed in pens (5 pens with 10 steers and 1 pen with 11 steers). Five steers in each pen were administered CCFA, and 5 served as control steers (1 pen had 6 control steers). The CCFA administration included a single-dose regimen (6.6 mg/kg, SC, on day 0), two-thirds-dose regimen (4.4 mg/kg, SC, on day 0), and 3-dose regimen (6.6 mg/kg, SC, on days 0, 6, and 13). Fecal samples were collected on days 0, 2, 6, 9, 13, 16, 20, and 28. Fecal samples were collected immediately before CCFA administration. Minimum inhibitory concentrations of 15 antimicrobials were determined for 3 E coli isolates/fecal sample. Escherichia coli were enumerated by use of direct-plating techniques. Results - Resistance to 1 or more antimicrobials was detected in 986 of 1,441 (68.4%) isolates recovered. Administration of CCFA was associated with a transient increase in the population of ceftiofur-resistant isolates. Susceptibility returned to day 0 values (ie, samples collected immediately before CCFA administration) approximately 2 weeks after completion of CCFA administration. Agreement between ceftiofur resistance and coresistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline was almost perfect (κ, 0.97). We did not detect variation in susceptibility of E coli recovered from commingled control steers. Conclusions and Clinical Relevance - Administration of CCFA provided selection pressure that favored transient expansion of multiple-resistant variants.