A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns is introduced, which can autonomously and selectively turn on the display of cytotoxic ligand patterns in tumour microenvironments. The switchable DNA origami normally hides six ligands but displays them as a hexagonal pattern 10 nm in diameter once under higher acidity. This can effectively cluster death receptors (DRs) and trigger apoptosis of human breast cancer cells at pH 6.5 while remaining inert at pH 7.4. When administered to mice bearing human breast cancer xenografts, this nanodevice decreased tumour growth by up to 70%. The data demonstrate the feasibility and opportunities for developing ligand pattern switches as a path for targeted treatment. Tumour-targeting strategies are plagued by the lack of ideal tumour-specific membrane receptors. The tumour necrosis factor (TNF) receptor superfamily (TNFRSF) plays essential roles in mammalian physiology. However, the ubiquitous expression of TNFRSF receptors on human cells prevents the development of biomolecules exclusively targeting specific cell populations via them. Nevertheless, ligands or antibodies of specific TNFRSF members have been developed to activate the apoptosis machinery of cancer cells. Recent studies, however, revealed that these ligands or antibodies lack efficacy due to their inability to precisely tune the oligomerization of the receptors on which the apoptosis cascades rely. By conjugating ligands to different scaffolds, an effective way to oligomerize certain members of TNFRSF can be achieved. Furthermore, to tune the inter-ligand distance with molecular precision, we and others have recently designed flat sheet-like DNA origamis for patterning ligands of death receptor 5 (DR5) or Fas receptors, revealing that hexagonal ligand patterns with specific inter-ligand distance at the nanoscale substantially drive apoptosis. However, these ligand patterns also interact with healthy cells. It is thus expected that, when applied in vivo, they risk promoting on-target, off-tumour toxicities, including neurotoxicity, increased susceptibility to infections and immune disorders. Cells of solid tumours exorbitantly consume oxygen and other nutrients, causing low oxygen tension and anaerobic glycolysis that ultimately leads to increased acidity. In this Article, to address the potential side effects of TNFRSF ligand patterns when used in vivo, we have developed a pH-sensitive three-dimensional DNA origami switch that can sense the high acidity of solid tumours. The device autonomously switches on surface display of six ligands as a sub-10 nm hexagonal pattern, while at higher pH this pattern remains hidden inside a cavity. The DNA origami switch is designed as an asymmetric double cylinder with a 24-nm-tall hollow head and a 15-nm-tall solid stem. The head has a 14-nm-deep cavity, where specific a. TheA DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns is introduced, which can autonomously and selectively turn on the display of cytotoxic ligand patterns in tumour microenvironments. The switchable DNA origami normally hides six ligands but displays them as a hexagonal pattern 10 nm in diameter once under higher acidity. This can effectively cluster death receptors (DRs) and trigger apoptosis of human breast cancer cells at pH 6.5 while remaining inert at pH 7.4. When administered to mice bearing human breast cancer xenografts, this nanodevice decreased tumour growth by up to 70%. The data demonstrate the feasibility and opportunities for developing ligand pattern switches as a path for targeted treatment. Tumour-targeting strategies are plagued by the lack of ideal tumour-specific membrane receptors. The tumour necrosis factor (TNF) receptor superfamily (TNFRSF) plays essential roles in mammalian physiology. However, the ubiquitous expression of TNFRSF receptors on human cells prevents the development of biomolecules exclusively targeting specific cell populations via them. Nevertheless, ligands or antibodies of specific TNFRSF members have been developed to activate the apoptosis machinery of cancer cells. Recent studies, however, revealed that these ligands or antibodies lack efficacy due to their inability to precisely tune the oligomerization of the receptors on which the apoptosis cascades rely. By conjugating ligands to different scaffolds, an effective way to oligomerize certain members of TNFRSF can be achieved. Furthermore, to tune the inter-ligand distance with molecular precision, we and others have recently designed flat sheet-like DNA origamis for patterning ligands of death receptor 5 (DR5) or Fas receptors, revealing that hexagonal ligand patterns with specific inter-ligand distance at the nanoscale substantially drive apoptosis. However, these ligand patterns also interact with healthy cells. It is thus expected that, when applied in vivo, they risk promoting on-target, off-tumour toxicities, including neurotoxicity, increased susceptibility to infections and immune disorders. Cells of solid tumours exorbitantly consume oxygen and other nutrients, causing low oxygen tension and anaerobic glycolysis that ultimately leads to increased acidity. In this Article, to address the potential side effects of TNFRSF ligand patterns when used in vivo, we have developed a pH-sensitive three-dimensional DNA origami switch that can sense the high acidity of solid tumours. The device autonomously switches on surface display of six ligands as a sub-10 nm hexagonal pattern, while at higher pH this pattern remains hidden inside a cavity. The DNA origami switch is designed as an asymmetric double cylinder with a 24-nm-tall hollow head and a 15-nm-tall solid stem. The head has a 14-nm-deep cavity, where specific a. The