Medium-pressure ion exchange chromatography was used for further fractionation of rice immunoactive peptides obtained from Trypsin hydrolyzation and gradient ethanol elution with DA201-C macroporous adsorption resin (40% ethanol fraction). The results showed that the optimal parameters for fractionation of RPHs-A3 fractions by WorkBeads 40 Q on a 2.6 cm×40 cm columns were as follows: Sample: 5 mL, 30 mg/mL 40% ethanol fraction; Equilibration buffer A: pH 9.0, 0.01 mol/L Tris-HCl; Elution Buffer B: pH 9.0, 0.01 mol/L Tris-HCl containing 1 mol/L NaCl; Sample flow rate: 1 mL/min; Elution flow rate: 10 mL/min. Five fractions were obtained after NaCl gradient elution. The results of MTT assay and LPS-stimulated RAW264.7 macrophages inflammation model showed that fraction RPHs-A3-B3 had the highest immune activity. RPHs-A3-B3 produced a significant effect on the proliferation of RAW264.7 macrophages with a stimulation index value of 1.315. Meanwhile, RPHs-A3-B3 significantly decreased the production of NO in LPS-stimulated RAW264.7 macrophages (P＜0.05). The inhibition rate was 8.28%. The research indicated that medium-pressure ion exchange chromatography can effectively fractionate rice immunoactive peptides. The SI value increased from 1.273 to 1.315. And RPHs-A3 had a significant inhibitory effect on intracellular NO release.