Objective To adapt the MDCK cells of adherent culture for suspension culture,establish and characterize a sus-pension cell line,so as to lay a foundation for the development of cell substrate-based influenza vaccines. Methods MDCK(NBL-2)cells of adherent culture were domesticated into suspension culture cells by shaker adaptation method,namedMDCK-S cells,and identified by 18S and species-specific PCR. The MDCK-S cells were subcultured to the 33rd passage asthe master cell bank(MCB),and passaged to the 38th generation as the working cell bank(WCB). MCB and WCB cellswere both detected for biological characteristics,contamination,tumorigenicity and oncogenicity,chromosome variability andinfluenza virus sensitivity,and observed under transmission electron microscope. Results The MDCK-S cells were confirmedto be dog-derived cells with no mutations and deletions,and there was no DNA contamination of other species. MCB andWCB cells showed good growth status,with“S”-shaped growth curves and doubling time of 27. 78 and 27. 21 h,respectively. Thecells were negative for bacteria,fungi and mycoplasma. The cell morphology,structure and cell membrane were intact,andthere were abundant microvilli on the membrane,indicating that the cell status was good,and there was little differencebetween different cell generations. MDCK-S cells formed epithelial-like subcutaneous tumors,demonstrating its tumorigenicitybut not oncogenicity. The proportion of chromosomes with 2 n = 78 ± 2 was approximately 86% to 96. 19%,and the numberof chromosomes remained stable from P29 to P50. In addition,MDCK-S cells were sensitive to influenza virus. Conclusion MDCK cells were successfully domesticated into suspension culture cells,and a suspension cell line MDCK-S was estab-lished in this study,which lays a foundation for the development of cell substrate-based influenza vaccines.