Stem cells are promising candidates for the regeneration of tissue and organ. We established the isolation and proliferation procedures involving multipotent stem cell derived from human subacromial bursal tissues and investigated the corresponding proliferative and differentiative characteristics. Human subacromial bursal tissue was obtained during arthroscopic rotator cuff surgery and single cells were acquired by dissociation with trypsin and ethylene diamine tetraacetic acid. Subacromial bursal tissue-derived colonies were Cultured in mesenchymal stromal cell growth medium. In passaged number 4-8, cultured fibroblast-like cells were harvested and marked with following antibodies and subjected to flow cytometry: CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD54, CD73 (SH3), CD90, CD105(SH2) and HLA class 1, and HLA-DR. Results: Human subacromial bursal tissue-derived colonies were highly proliferative and immunophenotypically-positive for CD13, CD29, CD44, CD54, CD73 (SH3), CD90, CD105(SH2) and HLA class 1, but negative for CD14, CD31, CD34, CD45 and HLA-DR. Incubation of these fibroblast-like cells with osteogenic agents resulted in the development of osteocytes. Under adipogenic conditions, lipogenous vacuoles were observed in the cells. Exposure of the cells to basic fibroblast growth factor resulted in neural differentiation. We established isolation and proliferation procedures for multipotent stein cells from human subacromial bursal tissues. Subacromial bursal tissue-derived multipotent stein cells expressed similar characteristics as mesenchymal stem cells. Considering the treatment for chronic large or massive rotator cuff tear, the effects of tridimensional matrices and scaffold for myogenic and fibrogenic induction of multipotent stem cells derived from subacromial bursal tissue, may be anticipated.