<p>This study evaluated the potential of <i>Levilactobacillus brevis</i> strains (20080 and G1) to alleviate intestinal inflammation and support gut health. Chronic inflammatory conditions are often associated with gut microbiota dysbiosis, suggesting a potential role for probiotics in its management. The anti-inflammatory effects of <i>L. brevis</i> strains were assessed in cellular models of systemic and intestinal inflammation using RAW 264.7 macrophages and HT-29 epithelial cells. In lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, <i>L. brevis</i> strains suppressed pro-inflammatory cytokines and nitric oxide (NO) production while enhancing anti-inflammatory responses. Furthermore, the expression levels of prostaglandin E₂ and leukotriene B4, which are key mediators of inflammatory diseases, particularly arthritis, were significantly reduced. Additionally, these strains effectively inhibited the activation of nuclear factor-κB, activator protein-1, and mitogen-activated protein kinase pathways, which are key regulators of inflammatory responses. In HT-29 cells, treatment with these strains led to a reduction in NO production upon sodium nitroprusside (SNP) stimulation. Under LPS stimulation, these strains suppressed the expression of pro-inflammatory cytokines and enhanced the expression of tight junction and mucin genes. In conclusion, <i>L. brevis</i> strains exhibited anti-inflammatory effects and gut-protective effects against intestinal inflammation, supporting their potential as therapeutic agents for inflammatory diseases.</p>