Co3O4 microspheres were synthesized in mass production by a simple hydrothermal treatment. One micrometer-sized spherical particles with well-crystallization could be obtained by XRD and SEM. Higher specific surface area (93.4m2 g−1) and larger pore volume (78.4cm3 g−1) by BET measurements offered more interfacial bondings for extra sites of Li+ insertion, which resulted in the anomalous large initial irreversible capacity and capacity cycling loss due to SEI film formation. The capacity retention of Co3O4 microspheres involved first forming acted as Li-ion anode material is almost above 90% from 12th cycle and it retain lithium storage capacity of 550.2mAhg−1 after 25 cycles, which show good long-life stability. The electrochemical impedance spectroscopy (EIS) tests before and after cyclic voltammetry measurements and charge–discharge experiments were carried out and the corresponding D Li values were also calculated. The relationship of the ac impedance spectra and the cycling behaviors was discussed. It is found that the decrease of capacity results from the larger Li+ charge-transfer impedance and the lower lithium-diffusion processes on cycling, which is in very good agreement with the electrochemical behaviors of Co3O4 electrode.