Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1alpha. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1alpha-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1alpha activity in hypoxia, in part through blocking MAPK activation.