Human hepatocarcinoma cells, HepG2 were cultured onto biodegradable polyglycolic acid (PGA) polymer scaffolds, which were cultured in a rotating cell culture system (RCCS) to form a three-dimensional (3D) multicellular culture in vitro. The RCCS can simulate microgravity effects with low shear stress and well exchanging for gas. Then the growth characteristics and some mechanism of the cells in RCCS were detected by scanning electron microscopy (SEM), transmission electron microscopy (TEM), RT-PCR and flow cytometry (FCM). The results indicate that the cells grew well with polyhedron morphology and lots of microvilli, mitochondria and tight junctions in this system, which means that this system is useful for cells to form 3D structure to mimic cell status in vivo. The expression of some cell adhesion molecules (CAMs) were changed markedly, which are closely associated with cancer invasion and metastasis. The characters of increased expression of integrin ?1(CD29), CD44, intercellular adhesion molecule-1(CD54) and depressed expression of E-cadherin presumably show that the HepG2 cells cultured in RCCS could recur some characters of primary liver cancer in vivo, the capacity of invasion and metastasis. It is necessary for acquiring perfect and external results to select an appropriate research model for studying in vitro. This 3D culture in vitro under simulated microgravity can provide a useful and reasonable model for oncology, anticancer drugs research and other research.