Aim We isolated Lactobacillus brevis G‐101 from kimchi lactic acid bacteria (LAB) strains, which induced IL‐10 expression in lipopolysaccharide (LPS)‐stimulated peritoneal macrophages. To evaluate the inflammatory effect of G‐101, we examined its inhibitory effect in 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitic mice. Materials and Results The colitic mice were prepared by intrarectal injection of TNBS. We measured intestinal mucosal cytokines by enzyme‐linked immunosorbent assay; activation of transcription factors, by immunoblotting; and macrophage polarization markers, by real‐time polymerase chain reaction. Of 200 LAB strains tested, Lact. brevis G‐101 showed most potent activity for induction of IL‐10 expression in LPS‐stimulated peritoneal macrophages. However, it significantly inhibited the expression of TNF‐α, IL‐1β and IL‐6 and the phosphorylation of IRAK1 and AKT, and activated NF‐κB and MAPKs. Treatment with TNBS caused colon shortening; increased myeloperoxidase activity; and increased IL‐1β, IL‐6 and TNF‐α expression in mice. Oral administration of Lact. brevis G‐101 significantly inhibited these activities. Lactobacillus brevis G‐101 inhibited TNBS‐induced IRAK‐1 phosphorylation and NF‐κB activation, as well as the expression of COX‐2 and iNOS. Lactobacillus brevis G‐101 inhibited the expression of M1 macrophage markers, but increased the expression of M2 macrophages in the colons of TNBS‐treated mice. Conclusions Lactobacillus brevis G‐101 may improve colitis by inhibiting the IRAK1/NF‐κB, MAPK and AKT pathways and by polarizing M1 macrophages to M2‐like macrophages. Significance and Impact of the Study These results suggest that IL‐10 expression‐inducing LAB can ameliorate colitis by inhibiting NF‐κB activation and macrophage polarization.