Spinel LiNi0.4Mn1.6O4 has been successfully synthesized by ultrasonic-assisted co-precipitation (UACP) method. The structure and physicochemical properties of this as-prepared powder compared with the LiNi0.4Mn1.6O4 synthesized by co-precipitation method were investigated by powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and galvanostatic charge–discharge test in detail. XRD and SEM show that all samples have high phase purity, and ultrasonic process plays an important role in controlling morphology; FT-IR reveals that the Mn(III)–O stretching band at 511cm−1 has a red shift to 503cm−1, and the Mn(IV)–O stretching band at 612cm−1 has a blue shift to 622cm−1 because of the doped Ni. CV confirms that the LiNi0.4Mn1.6O4 sample (UACP) has bigger area of the reduction peaks than that of sample synthesized by co-precipitation method, indicating that the former has higher discharge capacity than that of the latter. Galvanostatic charge–discharge test indicates that the initial discharge capacities for the LiNi0.4Mn1.6O4 (UACP) at C/5 and 1C are 129 and 116mAhg−1, respectively. After 100 cycles, their capacity retentions are 94.6% and 85.3%, respectively. EIS indicates that LiNi0.4Mn1.6O4 samples synthesized by UACP method have smaller charge transfer resistance than that of samples synthesized by co-precipitation method corresponding to the extraction of Li+ ions.