A total of 176 E. coli isolates were retrieved from 203 diarrheic fecal samples collected from Korean cattle on 117 different farms. The most frequently observed resistance in E. coli isolates was to tetracycline (88.6%), followed by streptomycin (80.7%) and ampicillin (64.8%). Resistance to cefazolin, cefoperazone, cefepime and amikacin was very low. Of the 176 E. coli strains, forty (22.7%) isolates from 30 farms showed resistance to fluoroquinolones (FQ). All the FQ-resistant strains possessed double mutations at codons 83 and 87 in the gyrA gene, and a single mutation mostly at codon 80 in the parC gene, except in one isolate. The pulsed-field gel electrophoresis profiles of the FQ-resistant E. coli isolates were heterogeneous, but two or three isolates that showed an identical pattern originated from the same or different farms. This study demonstrates that FQ resistance is frequently observed in E. coli from diarrheic cattle and that mutations in the quinolone resistance-determining region are the same as those seen in E. coli originating from other animal species and humans. The FQ resistance in diarrheic cattle might have been mostly acquired independently, although the possibility of transmission of FQ-resistant E. coli within a farm or between farms is plausible.