Complex spinel Li2CuTi3O8 with a space group of Fd-3m is prepared for the first time by a simple solid state reaction route. This compound has different space group from the previous reported Li2MTi3O8 (M = Zn, Co, Mg, Ni and Mn) with a space group of P4332. It shows reversibility for lithium storage in the cubic structure. A reversible capacity of 203 mAh g−1 can be delivered at a current density of 100 mA g−1 after 50 cycles with the capacity retention of 70.7%. The electrochemical reaction mechanism between Li2CuTi3O8 and lithium is investigated by various in-situ and ex-situ observations. Rietveld refinement results show that Li2CuTi3O8 has multiple interstitials to accommodate lithium ions during the lithiation process, in which the irregular octahedral 32e sites would be occupied by lithium ions at high working potentials and the regular octahedral 16c sites would be occupied by lithium ions at low working potentials. Besides, a reversible migration of copper ions can found during discharge process. Based on the reverse delithiation process, it is known that the whole charge–discharge process is quasi-reversible for Li2CuTi3O8. Therefore, Li2CuTi3O8 shows high structural stability as a promising lithium storage material for lithium-ion batteries.