BACKGROUND AND AIM: Formononetin (FMN) is a compound isolated from Astragalus membranaceus, that exhibits a range of pharmacological activities, including antitumor, anti-inflammatory, hypolipidemic, and antioxidant effects. Although preliminary study suggests that FMN have a therapeutic role in Inflammatory Bowel Disease (IBD), its specific mechanism of action requires further investigation. This study aimed to investigate the mechanism by which FMN treats DSS-induced colitis in mice. METHODS: RAW264.7 and Bone marrow-derived macrophages (BMDMs) were treated with LPS to establish an inflammatory cell model. Biochemical parameters and morphological characteristics were assessed in the present or absent of FMN. 4 % solution of DSS was administered to C57BL/6 mice to induce IBD, which served as an animal model for investigating the pharmacodynamics of FMN. RESULTS: FMN significantly reduced colitis-associated injury, as evidenced by a decrease in the disease activity index (DAI), weight gain, and restoration of colon length. Furthermore, FMN inhibits protein expression of NLRP3 inflammasome, suppressed the nuclear translocation of NF-κB/p65, and prevented mitochondrial damage, this process results in a reduction in the accumulation of reactive oxygen species (ROS). Additionally, FMN inhibited the mitogen-activated protein kinase (MAPK) signaling pathway, upregulated peroxisome proliferator-activated receptor gamma (PPAR-γ) in the nucleus, and decreased the release of inflammatory factors, thereby exerting anti-inflammatory effects. CONCLUSION: By inhibiting mitochondrial damage, activating the MAPK/PPAR-γ/ROS signaling pathway, reducing the nuclear translocation of NF-κB, and suppressing the expression of NLRP3 inflammasome-associated proteins, FMN exerts anti-inflammatory effects.