Abstract: OBJECTIVE To observe the prevalence of nanobacteria (NB) infection in bile and calculi of the patients with gallstones, analyze the correlation between the nanobacteria and formation of gallstone and explore the mechanisms of formation of gallstones so as to provide guidance for prevention and treatment of gallstones. METHODS The patients who received surgeries in Hangzhou Xixi Hospital from Oct. 2013 to Dec. 2014 were enrolled in the study.The bile specimens were collected from 43 patients including 36 gallstone patients and 7 non-gallstone patients and 30 patients with calculi during surgery. The bile and calculi specimens were cultured for NB by using cell culture method, and the NB strains were identified with the use of indirect immunofluorescence staining, transmission electron microscopy, alizarin red calcium staining and PCR. RESULTS Set the indirect immunofluorescence staining as the gold standard, the positive rate of NB was 44.18%(19/43) in the bile specimens, the positive rate of NB was 50.00%(18/36) in the bile specimens of the gallstone patients, 14.29%(1/7) in the bile specimens of the non-gallstone patients. The positive rate of NB was 60.00% in the calculi specimens; there was no significant difference in the positive rate of NB between the bile and calculi specimens of the gallstone patients. There was no significant difference in the detection of NB between the calcium staining method and the indirect immunofluorescence staining method and between the PCR and the indirect immunofluorescence staining method. CONCLUSION The NB is closely associated with gallstones. The incidence rate of NB infection is high among the gallstone patients, which may accelerate the formation of gallstones. The NB strains can be isolated from the bile and calculi specimens by cell culture and dilution double filtration method. Transmission electron microscopy and indirect immunofluorescence staining can be used as gold standards for identification of NB, and alizarin red calcium staining and PCR can provide significant reference.