The invention discloses a system for preparing deuterium-depleted water and deuterium-rich water by separating natural water. A core of the system is provided with a closed loop, a plurality of separation columns are sequentially connected in an end-to-end manner to form the closed loop, the system further comprises a raw material water storage tank, a deuterium-rich water storage tank, a deuterium-depleted water storage tank, a metering pump, a carburetor, pipelines and valves, and the pipelines and the valves are connected with the devices. According to the system, separation is implementedby the aid of moving bed simulation principles, raw material water vapor circularly moves in the closed loop formed by the separation columns through sequentially heating and cooling of the separationcolumns and control of a one-way valve, a constantly increased deuterium concentration gradient is acquired along with increase of cycle number, the deuterium-rich water is extracted from the top ofthe separation column at the near end of a heating loop, and the deuterium-depleted water is extracted from the bottom of the separation column at the far end of the heating loop after certain times of heating and cooling circulation of the separation columns are implemented. The system is simple in structure, convenient to control, smaller in device size and lower in construction cost, fillers are low in cost, and the natural water can be directly separated by the aid of a semi-continuous mode to prepare the deuterium-depleted water and the deuterium-rich water.