The therapeutic potential of mesenchymal stromal cells (MSCs) from various tissue origins have extensively been explored in both experimental and clinical studies, and peritoneal dialysis effluent-derived MSC (pMSC) may be an easily obtainable MSC source for clinical applications. In this study, we expanded and characterized the pMSCs after expansion in a human protein culture medium. The pMSCs were expanded in plastic dishes with the human protein medium. MSC marker expression was examined by flow cytometry. Spherical formation was tested by hanging drop method, and osteogenic, adipogenic, and chondrogenic differentiation capacities were confirmed by positive staining with Alizarin red, Oil red O, and Alcian blue, respectively. Here, we showed that after four passages of culturing in plastic dishes, pMSCs in the human protein medium displayed a homogeneous pattern of classical MSC markers (positive: CD29, CD44, CD73, CD90, and CD166; negative: CD14, CD34, CD45, CD79a, CD105, CD146, CD271, HLA-DR, SSEA-4, and Stro-1), while in the standard medium, pMSCs from some donors were CD45 or HLA-DR positive. For nonclassical MSC markers, pMSCs were CD200 positive from all the donors, negative for CD163, CD271, CD36, and CD248, and either positive or negative for CD274 and CD140b. Further, pMSCs from the human protein medium had the spherical formation capacity and multipotent differentiation capacity