In this work, three-dimensional mesoporous NiO nanostructures have been synthesized by a simple ethylene glycol (EG)-mediated self-assembly route and subsequent calcination process. The synthesized nickel alkoxide precursors annealed at 300 and 500°C exhibit different surface area, crystallinity and pore distribution, which have been characterized by scanning electron microscopy, transmission electron microscopy, selected area electron diffraction, X-ray diffraction, Fourier transform infrared spectroscopy and Nitrogen adsorption/desorption isotherms. The electrochemical properties of these NiO mesoporous nanostructures are investigated including the cycling and rate performance as anode materials for lithium-ion batteries. It is indicated that mesoporous NiO nanospheres synthesized at 500°C exhibit better electrochemical performance than that obtained at 300°C. The NiO nanospheres annealed at 500°C present a reversible specific capacity of 518mAhg−1 at a current density of 0.1Ag−1 after 60 cycles. With varying the rate from 0.1 to 8.0Ag−1, the capacity remains at 535mAhg−1 at 2Ag−1 after 30 cycles and resumes to 582mAhg−1 at 0.1Ag−1 after 60 cycles. The results indicate that our mesoporous NiO nanospheres are promising anode materials for lithium ion batteries.