AbstractThe old age‐related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T‐cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, thevis‐à‐visrole of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DCOld) or gut‐dysbiotic young (DCDysbiotic) but not young (DCYoung) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4+T cells. The mechanism deciphered for the loss of DCOldand DCDysbiotictolerance was chiefly through the overactivation of NF‐κB, impaired frequency of Tregs, upregulation in the level of pro‐inflammatory molecules (IL‐6, IL‐1β, TNF‐α, IL‐12, IFN‐γ), and decline in the anti‐inflammatory moieties (IL‐10, TGF‐β, IL‐4, IDO, arginase, NO, IRF‐4, IRF‐8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of theLactobacillusgenus was noticed in the gut. Replenishing the gut of old mice with theLactobacillus plantarumreinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age‐related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age‐associated disorders with theLactobacillus plantarum.