We showed previously that factor-inhibiting hypoxia-inducible factor HIF (FIH) monitors the expression of a spectrum of genes that are dictated by the cell's partial oxygen pressure. This action is mediated by the C-TAD, one of two transactivation domains (TADs) of the hypoxia-inducible factor. Here, we questioned: (1) the function of FIH as a HIF-1 modulator of gene expression in the context of a physiological oxygen gradient occurring in three-dimensional cultures and in tumors and (2) the role of FIH as a modulator of the growth of human tumor cells. We first showed that the expression pattern of HIF target genes that depend on the C-TAD, such as carbonic anhydrase IX, was spacially displaced to more oxygenated areas when FIH was silenced, whereas overexpression of FIH restricted this pattern to more hypoxic areas. Second, we showed that silencing fih severely reduced in vitro cell proliferation and in vivo tumor growth of LS174 colon adenocarcinoma and A375 melanoma cells. Finally, silencing of fih significantly increased both the total and phosphorylated forms of the tumor suppressor p53, leading to an increase in its direct target, the cell cycle inhibitor p21. Moreover, p53-deficient or mutant cells were totally insensitive to FIH expression. Thus, FIH activity is essential for tumor growth through the suppression of the p53-p21 axis, the major barrier that prevents cancer progression.