In bioinspired design, biological templates are mimicked in structure and function by highly controllable synthetic means. Of interest are static barrel‐like nanopores that enable molecular transport across membranes for use in biosensing, sequencing, and biotechnology. However, biological ion channels offer additional functions such as dynamic changes of the entire pore shape between open and closed states, and triggering of dynamic processes with biochemical and physical stimuli. To better capture this complexity, this report presents multi‐stimuli and mechano‐responsive biomimetic nanopores which are created with DNA nanotechnology. The nanopores switch between open and closed states, whereby specific binding of DNA and protein molecules as stimuli locks the pores in the open state. Furthermore, the physical stimulus of high transmembrane voltage switches the pores into a closed state. In addition, the pore diameters are larger and more tunable than those of natural templates. These multi‐stimuli‐responsive and mechanically actuated nanopores mimic several aspects of complex biological channels yet offer easier control over pore size, shape and stimulus response. The designer pores are expected to be applied in biosensing and synthetic biology. Biomimetic nanopores of complex function are built with DNA nanotechnology. The pores undergo dynamic changes in shape and can be locked in an open state by specific binding of DNA and protein stimuli, and close at high transmembrane potentials. The pores may be used in biosensing and synthetic biology.