Abstract: Objective To establish a stable method for the isolation, culture, and characterization of mouse lung- resident mesenchymal stem cells, which will then be used to study lung repair and regeneration mechanisms. Methods Mouse lungs were obtained via germ-free procedures and were mechanically minced and / or digested with collagenase. Cell morphology was observed by phase-contrast microscopy, cell surface markers were detected by flow cytometry, and cells were induced to differentiate into adipocytes or osteoblasts. Results We successfully isolated and cultured mouse lung- resident mesenchymal stem cells. FACS analysis demonstrated high expressions of Sca-1, CD90, CD29, and CD44, and low expressions of CD31 and CD45. The lung-resident stem cells differentiated into adipocytes or osteoblasts. Conclusions We isolated cells that expressed mesenchymal stem cell markers and had multi-directional differentiation ability.