Mammalian cells grow in three-dimensions (3-D) in vivo. Commonly used two-dimensional (2-D) cell cultures are inadequate to recreate the biological microenvironment of tumor cells. The potentially different outcomes from 2-D and 3-D culture systems may have a significant impact on the relevance of experimental findings. The purpose of this study was to characterize the human hepatoma cell line HepG2 in 2-D and 3-D cultures. HepG2 cells in 2-D and 3-D cultures were treated with cisplatin, 5-fluorouracil, and adriamycin and were analyzed by scanning electron microscopy and transmission electron microscopy. Cell cycle progression and apoptosis were detected by flow cytometry. Inhibition of cell proliferation was quantified by MTT assay. The expression of E-cadherin, CD44v6, VEGF, KDR, endostatin, Bax, and cytochrome-c were analyzed by immunohistochemical (IHC) staining. As compared to the 2-D monolayer culture, the 3-D multicellular spheroids (MCS) of HepG2 cells featured a greater fraction of cells in G1 phase and were organized with more abundant cell-cell adhesion. In addition, cells in MCS were significantly less apoptotic in maintenance culture media and were more resistant to drug-induced apoptosis. E-cadherin, CD44v6, VEGF, KDR, endostatin, and cytochrome-c levels were increased in MCS as compared to 2-D cell cultures. In conclusion, MCS conferred differentiated phenotypes including increased cell-cell adhesion and G1 phase cell cycle arrest, enhanced cellular resistance to apoptosis, and upregulated angiogenic potential. Based on our data, a multicellular morphological hierarchy may sustain the growth/survival advantages of cancer cells in vivo. Therefore, a 3-D culture system should be the preferred technique for cancer biology investigation.