The purpose of this study was to clarify the direct effect of hypoxia-inducible factor-1α (HIF-1α) on tumor growth, apoptosis and migration in vitro. To achieve this aim, a comparison was made of the differences in growth rates, apoptotic indices and cell invasive ability in the human cervical cancer cell line HeLa and the HIF-1α-blocked counterpart in a three-dimensional spheroid culture. A significant decrease in cell proliferation and invasion, and an increase in cell apoptosis were observed in HIF-1α-blocked cells in the three-dimensional culture. The data indicated that a multicellular spheroid culture is an ideal model of hypoxia in vitro and that HIF-1α is a significant regulator of adaptive processes that promote tumor cell malignant phenotypes, such as proliferation, anti-apoptosis and invasive ability.