Abstract: Manuka honey, known for its unique anti-inflammatory properties, has been extensively studied for its potential in the prevention and treatment of inflammatory diseases. To investigate the anti-inflammatory effects of Manuka honey, this study established inflammation models using HepG2 and Caco-2 cells induced by LPS. Cell viability and the expression of NF-κB signaling pathway genes were assessed through CCK-8, microscopic observation, RT-qPCR, and ELISA. The results showed that 1.0 μg/mL and 2.0 μg/mL LPS treatments caused significant nuclear deformation and disruption of tight junctions in HepG2 and Caco-2 cells. Despite these morphological changes, cell viability remained unaffected, and there was a significant upregulation in the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and COX-2) via the NF-κB signaling pathway. When the concentration of Manuka honey exceeded 20.0 mg/mL, a significant reduction in cell viability was observed. However, concentrations between 2.5–20.0 mg/mL of Manuka honey significantly inhibited the expression of these pro-inflammatory cytokines induced by LPS. Notably, the concentration of 10.0 mg/mL exhibited the best anti-inflammatory effect, with cytokine levels approaching the control group levels. This study suggests that Manuka honey has a significant inhibitory effect on inflammatory responses and can effectively alleviate LPS-induced cellular inflammation. It may serve as a potential adjunctive therapeutic strategy, offering new insights for the clinical treatment of inflammatory diseases.