A novel aryl hydrocarbon receptor nuclear translocator (ARNT)-interacting peptide (Ainpl) was characterized from human liver cDNA library using phage display. Ainpl suppresses hypoxia inducible factor-1a (HIF-1α) signaling pathway through an ARNTdependent manner. HIF-1α is known to be overexpressed in more than 90% of solid tumors, and the inhibition of HIF-1α is proved as an effective approach to suppress tumor growth. ARNT, as the obligatory heterodimeric partner of HIF-1α for downstream gene activation, was used as a bait to screen for Ainpl. Ainpl specifically interacts with the helix-loop-helix (HLH) subdomain of ARNT, but not with HIF-1α. GFP-Ainpl is localized in both cytoplasm and nucleus, and suppresses HIF-1α signaling by two mechanisms: (1) cytoplasmic GFP-Ainp 1 retains ARNT in the cell cytoplasm and (2) nuclear GFP-Ainpl inhibits HIF-1α/ARNT heterodimerization. The suppression of Ainpl on HIF-1α signaling was reversed by introducing ARNT into the cells using transient transfection. We further utilized HIV TAT protein transduction domain to deliver 6His-TAT-Ainpl into three different cancer cell lines (Hep3B, HeLa, MCF-7), and found that 6His-TAT-Ainpl co-localizes with ARNT in the cell nucleus. 6His-TATAinpl can be detected inside the cells after 30 min of transduction, and can reach the maximum level at 2 h. 6His-TAT-Ainp 1 remained detectable in the cells up to 96 h and had a half life of 24 h after transduction. In addition, 6His-TAT-Ainp 1 suppresses HIF-1α downstream genes at both message and protein levels in a dose-dependent manner. Taken together, molecules that target the HIF-1α and ARNT interface can be developed as viable drugs to suppress HIF-1α signaling.