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<i>Lespedeza bicolor</i> Turcz. (<i>L. bicolor</i>) honey, a monofloral honey, has garnered increased attention due to its origin in the <i>L. bicolor</i> plant. A previous study has shown that <i>L. bicolor</i> honey can ameliorate inflammation. In this study, we aimed to investigate the effects of <i>L. bicolor</i> honey extract and its biomarker (Trifolin) on DSS-induced ulcerative colitis (UC). Our results demonstrated that <i>L. bicolor</i> honey extract and Trifolin significantly increased the expression levels of the tight junction cytokines <i>Claudin-1</i> and <i>ZO-1</i>. Additionally, they decreased the pro-inflammatory factors <i>TNF-α</i> and <i>IL-6</i> and enhanced the antioxidant factors <i>NQO1</i> and <i>GSTA1</i>. Based on metabolomic analyses, <i>L. bicolor</i> honey extract and Trifolin regulated the progression of UC by inhibiting ferroptosis. Mechanistically, they improved the levels of SOD and iron load, increased the GSH/GSSG ratio, reduced MDA content and ROS release, and upregulated the <i>Nrf2</i>/<i>HO-1</i> pathway, thereby inhibiting DSS-induced UC. Moreover, the expression levels of ferroptosis-related genes indicated that they decreased <i>FTL</i>, <i>ACSL4</i>, and <i>PTGS2</i> while increasing <i>SLC7A11</i> expression to resist ferroptosis. In conclusion, our study found that <i>L. bicolor</i> honey improves DSS-induced UC by inhibiting ferroptosis by activating the <i>Nrf2</i>/<i>HO-1</i> pathway. These findings further elucidate the understanding of anti-inflammatory and antioxidant activities of <i>L. bicolor</i> honey.