We have investigated for the first time the structure of i-motif DNA in solution at various pH conditions by using synchrotron small-angle X-ray scattering technique. To facilitate direct structural. comparison between solution structures of i-motif DNA at various pH values, we created atomic coordinates of i-motif DNA from a fully folded to unfolded atomic model. Under mild acidic conditions, the conformations for i-motif DNA appeared to be similar to that of the partially unfolded i-motif atomic model in overall shape, rather than the fully folded i-motif atomic model. Collectively, our observations indicate that i-motif DNA molecule is structurally dynamic over a wide pH range, adopting multiple conformations ranging from the folded i-motif structure to a random coil conformation. As the i-motif structure has been used as an important component in nanomachines, we can therefore believe that the structural evidence presented herein will promote the development of future DNA-based molecular-actuator devices.