Hypoxia,　a　ubiquitous　hallmark　in　cancer,　underscores　the　significance　of　targeting　HIF-1α,　the　principal　transcriptional　factor　of　hypoxic　responses,　for　effective　cancer　therapy.　Herein,　DNA　yokes,　a　novel　class　of　DNA　nanomaterials　harboring　specific　HIF-1α　binding　sequences　(hypoxia　response　elements,　HREs),　are　introduced　as　nanopharmaceuticals　for　cancer　treatment.　Comprising　a　basal　tetrahedral　DNA　nanostructure　and　four　HRE-bearing　overhanging　chains,　DNA　yokes　exhibit　exceptional　stability　and　prolonged　intracellular　retention.　The　investigation　reveals　their　capacity　to　bind　HIF-1α,　thereby　disrupting　its　interaction　with　the　downstream　genomic　DNAs　and　impeding　transcriptional　activity.　Moreover,　DNA　yokes　facilitate　HIF-1α　degradation　via　the　ubiquitination　pathway,　thereby　sequestering　it　from　downstream　targets　and　ultimately　promoting　its　degradation.　In　addition,　DNA　yokes　attenuate　cancer　cell　proliferation,　migration,　and　invasion　under　hypoxic　conditions,　while　also　displaying　preferential　accumulation　within　tumors,　thereby　inhibiting　tumor　growth　and　metastasis　in　vivo.　This　study　pioneers　a　novel　approach　to　cancer　therapy　through　the　development　of　DNA-based　drugs　characterized　by　high　stability　and　low　toxicity　to　normal　cells,　positioning　DNA　yokes　as　promising　candidates　for　cancer　treatment.　©　2024　Wiley-VCH　GmbH.