: Objective: This study aims to explore the effect of hydroxychloroquine(HCQ)on dextran sulfate sodium salt(DSS)-induced colitis in mice and to reveal the underlying mechanisms of HCQ in the inhibition of M1 macrophages polarisation.Methods: All the C57BL/6 mice were randomly divided into 3 groups: the control group (common drinking water+200 μL pure water by gavage), DSS group (3.5% DSS drinking water+200 μL pure water by gavage) and DSS+HCQ group (3.5% DSS drinking water+200 μL HCQ of 60 mg/kg by gavage). The disease activity index(DAI)score was evaluated according to stool property and body weight change every day. The mice in each group were sacrificed after modeling experiment with the colon length measured. The colon tissues were stained with haematoxylin and eosin (H E) for histological score calculation. The colonic lamina propria mononuclear cells (LPMCs) were isolated and the proportion of M1 macrophages was measured by flow cytometry. In vitro, bone marrow-derived macrophages (BMDMs) were isolated and stimulated to induce M1 macrophages with the proportion measured by flow cytometry. The levels of p-STAT1, IRF5, NF-κB /p65,etc in the macrophages were detected by western blotting. Results: HCQ ameliorated DSS-induced colitis and significantly decreased the percentage of M1 macrophages in colonic LPMCs in DSS-induced colitis mice. In vitro, HCQ significantly repressed M1 polarisation. HCQ decreased the phosphorylation of NF-κB and the downstream of NLRP3 inflammasome formation in M1 macrophages. The similar results were observed with PDTC treatment (an inhibitor of NF-κB signaling). Conclusion: HCQ attenuated colonic inflammation in mice by supressing M1 macrophage polarization through the inhibition of NF-κB/NLRP3 signaling pathway.