The lithium-ion batteries with high energy density, large power density and long cycle life have attracted much attention. Among various anodes, the CuO anode possesses a high specific capacity of 670 mAh g(-1). However, the CuO anode suffers from capacity fading after the first cycle, and the reason is not clear up enough to now. To investigate the nature of CuO anode lithiation-delithiation conversion in lithium-ion batteries, we investigated the phase conversion of CuO anode by in situ X-ray diffraction. We discovered that the specific discharge capacity of CuO during the first discharge cycle is usually more than 670 mAh g(-1), but this value decreases to approximately 400-450 mAh g(-1) after the second cycle and is stable for the subsequent cycles. The in situ X-ray diffraction observations reveal that in the discharge process of the first cycle, the CuO crystalline phase changes gradually to Cu. However, during the charge process of the first cycle, the delithiation product phase is Cu2O. The same phenomenon occurs again in the second charging-discharging cycle. The obtained scientific understanding reveals that the nature of CuO anode lithiation-delithiation phase conversion in Li-ion battery is between Cu2O and Cu, not CuO and Cu, which is responsible for capacity fading of CuO anode. The present research provides a certain scientific basis for the application of CuO in lithium-ion batteries. The in situ XRD method is helpful to understand the mechanism of electrochemical behaviour of other transition metal oxygen (sulphur, and fluorine) and can make an in-depth study of the searching for commercial anode materials for lithium-ion batteries.