Objective To investigate the lung transplantation by establishing an orthotopic mouse lung transplantation model by microsurgery. Methods Thirty SPF male C57BL/6 mice served as both recipient and donor. Orthotopic lung transplantation models were established in the mice by three-cuff anastomotic technique. Native and transplanted lung tissue samples were taken at 7, 14, 21 and 28 days after transplantation for pathological evaluation of the outcomes using HE staining. Results After learning curve, 30 mice received transplantation. All mice survived and the success rate of lung transplantation was 89%. The donor operation time was 35.2±9.81 min and recipient operation time was 24.6±7.42 min. Mean cold ischemia time was 46.6±8.92 min, and warm ischemia time was 17.2±3.08 min. No histological alterations were routinely detected in the isograft lungs at all times post-transplantation compared with the native lungs. Conclusions Mouse lung transplantation model can be successfully established by the established technique with a high success rate and excellent reproducibility, and is in accordance with the physiology of clinical orthotopic lung transplantation. It is a good animal model for studying the pathogenesis and treatment of lung transplantation.