AbstractThis study aimed to explore the effect and the molecular mechanism of tetrahedral framework nucleic acids (tFNAs), a novel self‐assembled nanomaterial with excellent biocompatibility and superior endocytosis ability, in inhibition of pathological retinal neovascularization (RNV) and more importantly, in amelioration of vaso‐obliteration (VO) in ischaemic retinopathy. tFNAs were synthesized from four single‐stranded DNAs (ssDNAs). Cell proliferation, wound healing and tube formation assays were performed to explore cellular angiogenic functions in vitro. The effects of tFNAs on reducing angiogenesis and inhibiting VO were explored by oxygen‐induced retinopathy (OIR) model in vivo. In vitro, tFNAs were capable to enter endothelial cells (ECs), inhibit cell proliferation, tube formation and migration under hypoxic conditions. In vivo, tFNAs successfully reduce RNV and inhibit VO in OIR model via the PI3K/AKT/mTOR/S6K pathway, while vascular endothelial growth factor fusion protein, Aflibercept, could reduce RNV but not inhibit VO. This study provides a theoretical basis for the further understanding of RNV and suggests that tFNAs might be a novel promising candidate for the treatment of blind‐causing RNV.