LiNi0.5Mn1.5O4 thin films were coated on stainless steel substrates by radio-frequency sputtering at room temperature for a lithium-ion battery. The cathode films were post-annealed at 500, 600 and 700 °C to study (i) the impact of annealing temperature on the crystallization of the cathode film and (ii) the reactivity between the cathode film and the stainless steel substrate. X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy were adopted to characterize the thin films. The electrochemical properties of the LiNi0.5Mn1.5O4 cathodes were investigated with cyclic voltammetry and galvanostatic charge/discharge tests. As the annealing temperature increased from 500 to 600 °C, the crystallinity and electrochemical characteristics of LiNi0.5Mn1.5O4 both improved. However, as the annealing temperature further increased to 700 °C, the phase purity and the electrochemical performance were greatly deteriorated. Combined chemical analyses with time-of-flight secondary ion mass spectrometry depth profiling and energy-dispersive X-ray spectroscopy in scanning transmission electron microscopy mapping have indicated that the cross-diffusion of metal ions between LiNi0.5Mn1.5O4 and the stainless steel substrate takes place at a temperature higher than 600 °C, which was attributed as a main origin of the phase change in the LiNi0.5Mn1.5O4 cathode layer and electrochemistry degradations of lithium-ion battery.