Objective To investigate the mechanisms of both levofloxacin and ciprofloxacin resistance in clinical strains of Pseudomonas aeruginosa isolated from our hosptial.Methods Twenty P .aeruginosa isolates resistant to both levofloxacin and ciprofloxacin as tested by VITEK-2 Compact were collected.Agar dilution method was used to confirm their minimum inhibito-ry concentrations (MICs)of levofloxacin and ciprofloxacin.DNA gyrase (gyrA and gyrB )and topoisomerase IV (parC and parE)were analyzed by PCR amplification.The expression of efflux systems were analyzed by real-time RT-PCR.Results The MIC results were consistent between Agar dilution method and Vitek-2 Compact system.DNA gyrase (gyrA and gyrB )se-quencing analysis showed that the mutations were mainly frame-shifting mutation characteristic of base (C)insertion at position 941 in gyrA gene,deletion of base (A)at position 1588 in gyrB gene,and insertion of base (T)at position 1543 in gyrB gene.Insertion of base (C)at position 1895 in parE gene was identified in 3 strains.Overexpression of MexAB-OprM and MexCD-OprJ was detected by real-time RT-PCR.Conclusions The insertion and/or deletion of bases in DNA gyrase (gyrA and gyrB)genes and overproduction of MexAB-OprM and MexCD-OprJ efflux systems may contribute to the resistance to both ciprofloxacin and levofloxacin in clinical isolates of P .aeruginosa.