Abstract Membrane‐penetrating molecular devices are valuable biological tools. Herein, we describe membrane‐targeting molecular devices based on the triplexes of poly T acyclic L‐threoninol nucleic acid (L‐ a TNA) tethered site‐specifically to cholesterol and poly A DNA. The L‐ a TNA was linked to cholesterol in two different orientations. The membrane‐binding properties differed depending on the orientation at the cholesterol linkage. One triplex adhered to giant unilamellar vesicles and the other penetrated these vesicles. When the membrane‐penetrating triplex was formed with a photoresponsive DNA, photoresponsive signal transduction into giant unilamellar vesicles was enabled. These membrane‐targeting molecular devices have potential as biosensors, artificial organs, and molecular robots and in chemical artificial intelligence. Moreover, this strategy for post‐modification of the propargyl‐L‐ a TNA unit will enable further functionalization of L‐ a TNA and other acyclic XNAs.