Inflammation is a critical immune response to harmful stimuli such as infections or injuries. However, excessive inflammation can lead to tissue damage, chronic diseases, and systemic inflammatory response syndrome, resulting in multiple organ dysfunction. This dysregulated response can trigger a massive inflammatory cascade, which may prove fatal. Current anti-inflammatory treatments can alleviate symptoms but often have adverse effects, including gastrointestinal and cardiovascular complications. Tragopogon dubius Scop. (T. dubius) is a flowering plant from the Asteraceae family with edible stems. Despite reports of its effectiveness against periodontitis, overall bioactivity beyond this application remains insufficiently explored. This study investigates the anti-inflammatory properties and underlying mechanisms of T. dubius methanolic extract in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and ICR mice. The methanolic extract of T. dubius inhibited LPS-stimulated inflammatory mediator nitric oxide (NO) without exhibiting cytotoxicity under 1,000 μg/mL. It also decreased the expression of inducible NO synthase (iNOS) and interleukin-6 (IL-6) at both mRNA and protein levels. Additionally, the T. dubius methanolic extract inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) in LPS-stimulated RAW 264.7 macrophages. Furthermore, T. dubius methanolic extract mitigated weight loss and significantly improved survival rates in the LPS-induced inflammatory animal model. It also reduced the levels of NO and IL-6 in the plasma. Overall, the findings suggest that T. dubius methanolic extract has significant anti-inflammatory properties, primarily through inhibiting the JNK/NF-κB signaling pathway.