NiO microspheres with well-defined hollow structure were successfully obtained using spherical yeast as a natural bio-template. The as-prepared samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The morphologically uniform NiO hollow microspheres prepared with yeast template after calcination had an averaged diameter of 1.5–2 μm and the shell thickness of ∼500 nm. The concentration of Ni(NO3)2, calcination temperature and aging time play important roles on the crystal phase and morphologies of the NiO hollow microspheres. Meanwhile, the NiO/C hollow microspheres were successfully fabricated via a simple hydrothermal method using glucose as carbon source. A possible formation mechanism of forming NiO/C hollow microspheres was proposed and the electrochemical performance of the NiO/C hollow microspheres as anode material for Li-ion batteries was investigated. Compared with the samples prepared without template or without carbon coating, the as-prepared NiO/C hollow microspheres show higher discharge capacity and better cycle performance as an anode material in Li-ion batteries.