Hypoxia-inducible factor 1 (HIF-1), a master heterodimeric transcriptional regulator consisting of HIF-1alpha and HIF-1beta subunits for cellular response to hypoxia, plays an important role in carcinogenesis, while CCAAT/enhancer-binding protein alpha (C/EBPalpha) is proposed to act as a tumor suppressor in C/EBPalpha-expressing tissues. Previously, we reported that ectopically expressed HIF-1alpha protein interacts with and enhances transcriptional activity of C/EBPalpha, which favors leukemic cell differentiation. Here we further showed that such an interaction also occurred in their endogenously expressing state of leukemic U937 cells. Glutathione S-transferase pull-down assay proposed that the protein-protein interaction was direct, and transactivation domains of C/EBPalpha and the basic helix-loop-helix domain of HIF-1alpha were essential for such an interaction. More intriguingly, we provided the first demonstration that C/EBPalpha competed with HIF-1beta for direct binding to HIF-1alpha protein. Correspondingly, C/EBPalpha overexpression significantly inhibited the DNA-binding ability of HIF-1 and expressions of hypoxia-responsive element-driven luciferase and HIF-1-targeted genes vascular endothelial growth factor, glucose transporter-1 and phosphoglycerate kinase 1. In parallel, suppression of C/EBPalpha expression by specific small hairpin RNA increased DNA-binding ability of HIF-1 and expression of these HIF-1-targeted genes in leukemic U937 cells. These results would provide new insights for antitumor potential of C/EBPalpha protein.