CD44, a cell-surface glycoprotein, plays an important role in cell proliferation, adhesion, migration, and other biological functions, which are related with the physiological and pathologic activities of cells. Especially, CD44 is extensively expressed within adult bone marrow and has been considered as an important marker for some cancer stem cells (CSCs) in various types of tumors. Therefore, it is essential to understand the variations in CD44 expression of stem cells and cancer cells for further clinical applications. In this paper, CD44 expression was assessed on a human colon cancer cell line (SW620), a human mesenchymal stem-like cell line (3A6), and a human foreskin fibroblast line (Hs68). We used chitosan to establish a suspension culture model to develop multicellular spheroids to mimic a three-dimension (3D) in vivo environment. Obviously, CD44 expression on 3A6 and SW620 cells was dynamic and diverse when they were in the aggregated state suspended on chitosan, while Hs68 cells were relatively stable. Furthermore, we discuss how to regulate CD44 expression of 3A6 and SW620 cells by the interactions between cell and cell, cell and chitosan, as well as cell and microenvironment. Finally, the possible mechanism of chitosan to control CD44 expression of cells is proposed, which may lead to the careful use of chitosan for potential clinical applications.