Background: Sulfonamides is the second most popular antibiotic in many countries, which leads to the widespread emergence of sulfonamides resistance. Sul3 is a late sulfanilamide resistance gene, whose research is relatively little. Result: 46 sul3 positive E. coli strains were separated. A total of 12 ST types were observed, and 1 of those was previously unknown type. The ST350 is the most numerous type. All isolates were multidrug-resistant E. coli, with high antimicrobial rates to penicillin, ceftriaxone sodium, streptomycin, tetracycline, ciprofloxacin, gatifloxacin and chloramphenicol (100%, 73.9%, 82.6%, 100%, 80.4%, 71.7% and 97.8%), and with at least 3 resistance genes in addition to sul3. The plasmids transfered from 3 sul3-positive isolates to C600, the most of which brought 7 antibiotic resistance and increased resistance genes to C600. The transferred sul3 gene and the plasmid that carries it could be stably inherited in the recipient bacteria for at least 20 days. Those plasmids had no effect on the growth of the recipient bacteria, but it would greatly reduce (at least 60 time) the in vitro competitiveness of the strains. Conclusions: In Nanning, these sul3-positive Escherichia coli have strong antimicrobial resistance, and the plasmid carrying sul3 has the ability to transfer multiple resistance genes, so long-term monitoring is necessary. Since the transferred plasmid will greatly reduce the in vitro competitiveness of the strain, we can consider limiting the spread of antimicrobial in this respect.