A fundamental physiological property of mammalian cells is the regulation of proliferation according to O₂ availability. Progression through the cell cycle is inhibited under hypoxic conditions in many, but not all, cell types, and this G1 arrest is dependent on hypoxia-inducible factor (HIF) 1α. Components of the hexameric MCM helicase, which binds to replication origins before the onset of DNA synthesis, are present in large excess in mammalian cells relative to origins, suggesting that they may have additional functions. Screens for HIF-1α interacting proteins revealed that MCM7 binds to the amino-terminal PER-SIM-ARNT (PAS) domain of HIF-1α and stimulates prolyl hydroxylation-dependent ubiquitination and degradation of HIF-1α, whereas MCM3 binds to the carboxyl terminus of HIF-1α and enhances asparaginyl hydroxylation-dependent inhibition of HIF-1α transactivation domain function. Thus MCM proteins inhibit HIF activity via two distinct O₂-dependent mechanisms. Under prolonged hypoxic conditions, MCM mRNA expression is inhibited in a HIF-1α-dependent manner. Thus HIF and MCM proteins act in a mutually antagonistic manner, providing a novel molecular mechanism for homeostatic regulation of cell proliferation based on the relative levels of these proteins.