To investigate the reparative effects of Lacticaseibacillus rhamnosus, mediated by mulberry galacto-oligosaccharide (MGO), on inflammatory cells. This study utilized a lipopolysaccharide (LPS)-induced inflammatory model in HT-29 cells and employed the CCK-8 assay, enzyme-linked immunosorbent assay, real-time quantitative PCR, and Western blot analysis for evaluation. The results showed that following treatment with 1.00 μg/mL LPS, the secretion and mRNA levels of pro-inflammatory factors in HT-29 cells increased significantly, and iNOS, COX-2, IL-1β, and NF-κB p65 were upregulated significantly (P < 0.05). When Lacticaseibacillus rhamnosus at a concentration of 10_⁴ CFU/mL was administered as an intervention, and a 30 μg/mL dexamethasone solution was used as a positive control, the expression of LPS-induced pro-inflammatory cytokines was significantly inhibited. Treatment with MGO-mediated Lacticaseibacillus rhamnosus produced the best anti-inflammatory effects, with a significant reduction in inflammatory cytokine levels compared to the model group. Its anti-inflammatory efficacy was comparable to that of dexamethasone. This study suggests that MGO-mediated Lacticaseibacillus rhamnosus exerts an inhibitory effect on inflammatory responses and can effectively suppress NF-κB signaling and reducing pro-inflammatory mediators, it effectively alleviates LPS-induced inflammatory damage in HT-29 cells. This provides new insights for the resource development of mulberry synbiotics and the clinical treatment of inflammatory bowel diseases.