Hypoxia is a common environmental stress that influences signaling pathways and cell function, which through initiates intracellular signaling pathways and hence leads to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). In this study, we initially confirm that hypoxia activates HIF-1α protein expression in a time-dependent manner with a maximum reached at 60 min in vitro and 4h in vivo in gastric mucosa epithelial cells. The expression of HIF-1α is correlated with the activation of HIF-1 DNA binding and transcriptional activity. Hypoxia does not affect HIF-1α mRNA transcription but regulates HIF-1α protein expression through a translation-dependent pathway to regulate protein synthesis. Hypoxia could induce phosphorylation of Akt, MAPK (ERK), and a target of p70S6K1. PI3K and MAPK inhibitor and LY294002 and U0126 could inhibit hypoxia-induced HIF-1 and VEGF expression. We also investigated the role of reactive oxygen species (ROS) involved in HIF-1 and VEGF expression. Exogenous addition of H2O2 was sufficient to activate Akt and ERK, scavengers of H2O2 significantly inhibited hypoxia-induced Akt and ERK, and subsequent HIF-1α expression and transcriptional activity. In conclusion, our data suggested that hypoxia-PI3K signaling through Akt and ERK kinases regulated ROS-dependent, hypoxia-induced HIF-1 activation and VEGF expression in gastric mucosa epithelial cells.