Abstract: An in vitro gut simulation model has been developed to study the effects of low doses of ciprofloxacin on human Escherichia coli and Enterococcus faecalis. The ciprofloxacin-resistance-determining regions (QRDR) of gyrA in the ciprofloxacinresistant bacteria were amplified by polymerase chain reaction in order to study the mechanism of antibiotic resistance. The result showed that Escherichia coli could produce antibiotic resistance to ciprofloxacin, but not to other antibiotics. Enterococcus faecalis did not produce antibiotic resistance to all antibiotics. Those ciprofloxacin-resistant Escherichia coli strains had double mutations of C-to-T transition at position 248 (Ser 83 to Leu) and G-to-T transition at position 259 (Asp 87 to Tyr). The study demonstrated that low concentration of ciprofloxacin had selective pressure on human intestinal flora and could induce Escherichia coli to produce antibiotic resistance. It supplies the data for safety assessment on ciprofloxacin residues in foods of animal origin.