Hepatocellular carcinoma (HCC) treatments are evaluated by two‐dimensional (2D) in vitro culture systems, despite their limited ability to predict drug efficacy. The three‐dimensional (3D) microporous scaffold provides the possibility of generating more reliable preclinical models to increase the efficacy of cancer treatments. The physical properties of a microporous cellulosic scaffold were evaluated. The cellulosic scaffold was biocompatible and had a highly porous network with appropriate pore size, swelling rate, and stiffness of cancer cell cultures. Cellulosic scaffolds were compared with 2D polystyrene for the culture of HepG2 and Huh7 human HCC cells. Cellulosic scaffolds promoted tumor spheroid formation. Cells cultured on scaffolds were more resistant to chemotherapy drugs and showed upregulation of EpCAM and Oct4. The migration ability of HCC cells cultured on scaffolds was significantly greater than that of cells grown in 2D cultures as evidenced by the downregulation of E‐cadherin. In addition, the proportion of CD44+/CD133+ HCC cancer stem cells (CSCs) was significantly greater in cells cultured on scaffolds than in those grown in 2D cultures. These findings suggest that cellulosic scaffolds effectively mimic the in vivo tumor behavior and may serve as a platform for the study of anticancer therapeutics and liver CSCs.