The cycle life of Li4Ti5O12-based lithium ion batteries (LIBs) is closely related to the quality of the protective passivation film formed on Li4Ti5O12 anode, which is elaborately constructed during formation process. In this work, the passivation film formation on Li4Ti5O12 anodes during Li+ intercalation/deintercalation processes and at different lithiation states is investigated. Passivation film formed in the lithiation state of Li4Ti5O12 is more efficient than delithiation state. The film formation is related to the concentration of Ti3+ active sites on the surface of Li4Ti5O12 particles. There is an optimum range of lithiation degree of Li4Ti5O12 for the completion of uniform passivation film formation on the electrodes. Excessively high lithiation state is proved unnecessary or even detrimental for effective surface layer construction. Compared with traditional formation protocols with fully charge-discharge cycles, simply storing Li4Ti5O12-based LIBs in the optimized lithiation range at high temperature can construct efficient and stable passivation film. Such a formation protocol can greatly reduce the cost and simplify the manipulation process of Li4Ti5O12-based batteries for the LIBs enterprises.