Aim To investigate the effect of probucol on the differentiation of inflammatory monocyte subsets and its potential mechanism. Methods The primary single cell suspension of spleen was prepared from 8 week old C57BL/6 mice. The differentiation of monocyte subsets was induced by 100 kU/L recombinant interferon-γ, then intervened with 5,0, 100 μmol/L probucol, in order to observe their effects on the differentiation of monocytes. The cells were stained with anti-CD11b-PE and anti-Ly-6C-FITC antibody, and the differentiation of monocyte subsets was detected by flow cytometry, then the data were analyzed by Flowjo software. Using intracellular cytokine staining and 2′,7′-dichlorofluorescein-diacetate probe, the level of reactive oxygen species (ROS) in monocyte subsets was detected by flow cytometry. NADPH oxidase activity in human monocytic cell line THP-1 was detected by using NADPH oxidase test kit. Results In a concentration dependent manner, 5,0, 100 μmol/L probucol inhibited the differentiation of Ly-6C^hi inflammatory monocyte subsets in the spleen primary cells. The level of ROS in Ly-6C＋ inflammatory monocyte subset was 2 times of that in Ly-6C－ inflammatory monocyte subset. At the same time, 5,0, 100 μmol/L probucol inhibited the production of ROS in Ly-6C＋ inflammatory monocyte subset in a concentration dependent manner. 100 μmol/L probucol significantly inhibited the activity of NADPH oxidase in THP-1 cells. Conclusion Probucol may interfere with the activity of NADPH oxidase, thereby inhibiting the differentiation of Ly-6C^hi inflammatory monocyte subset and the production of ROS.