Nickel oxide deposited silica stationary phase was prepared by liquid phase deposition and characterized. Its chromatographic performance was evaluated using several compounds such as typical polar compounds and benzimidazoles as probes. The effects of mobile phase variables such as acetonitrile content, salt concentration, and pH on the chromatographic behavior, thermodynamics, and kinetics of these compounds were investigated to reveal the retention mechanism. The results showed that the prepared stationary phase exhibited a retention behavior of hydrophilic interaction liquid chromatography, and that multiple retention mechanisms including partitioning, adsorption, ionexchange, electrostatic attraction, and coordination interactions contributed to solute retention. The coordination of nickel oxide electron-accepting sites and the electron-donating solutes resulted in the mixed-mode retention on stationary phase, which could be very useful for enhancing the chromatographic selectivity for the analytes. The batch-to-batch reproducibility was acceptable with the relative standard deviations of probe retentionof less than 9.89%. The prepared nickel oxide deposited silica stationary phase was successfully employed for the separation of several compounds, and it showed better separation effect and different selectivity from silica column and commercial Zorbax NH 2 column.