The present work illustrates unique design, construction and operation of an i-motif-based DNA nanomachine templated on gold nanoparticles (AuNPs), which utilizes pH-responsive dynamic motion of i-motif DNA strands and aggregational behavior of AuNPs to elicit programmed delivery of therapeutic siRNA. The pH-sensitive nucleic acids immobilized on the AuNPs consisted of three functional segments, i.e., an i-motif DNA, an overhanging linker DNA and a therapeutic siRNA. At neutral pH, the i-motif DNA is hybridized with the overhanging linker DNA segment of the therapeutic siRNA. However, in endosomal acidic pH, the i-motif DNA forms interstrand tetraplex, which could induce cluster formation of AuNPs resulting in endosomal escape of AuNP clusters, and produce a high gene silencing efficiency by releasing siRNA in the cytosol. Furthermore, the cluster formation of AuNPs accelerated photothermal ablation of cells when irradiated with laser. Precise and synchronized biomechanical motion in subcellular microenvironment is realized through judicious integration of pH-responsive behavior of the i-motif DNA and AuNPs, and meticulous designing of DNA.