Objective To determine the effect of pH value on the drug resistance and biofilm formation of Klebsiella pneumoniae (K. pneumoniae) strains isolated from respiratory tract in order to provide the theoretical reference for clinical anti-infective treatment. Methods Twelve strains of K. pneumoniae (named as K1 to K12) were isolated from the sputum samples in the patients hospitalized in the Department of Respiratory Diseases of our hospital. At different pH values (5.5,6.5 and 7.5), the minimal inhibitory concentration (MIC) of the 12 strains was determined respectively using the broth microdilution, the adhesion of K. pneumoniae was measured by flat colony counting, and the biofilm formation was observed with 96-well crystal violet staining. Clinical data were analyzed by SPSS statistics 17.0. The measurement data were expressed as mean±standard deviation ([AKx-D]±s), and Student’s t test was used for comparison between groups. Results With the decrease of pH value, the MIC of ceftazidime to K. pneumoniae was decreased, whereas that of levofloxacin was increased. However, there was no correlation of the effects of pH value with the MIC of gentamicin. Compared with pH 5.5 value, the adhesion and biofilm formation were increased significantly in most K. pneumoniae isolates at pH 6.5 (P<0.05), while were enhanced remarkably in all the strains at pH 7.5 (P<0.05). Conclusion Different pH values affect the drug resistance and biofilm formation of K. pneumoniae strains.