Background and Aim Nuclear factor kappa B (NF‐κB) activation and endoplasmic reticulum (ER) stress signaling play significant roles in the pathogenesis of inflammatory bowel disease (IBD). Thus, we evaluated whether new therapeutic probiotics have anti‐colitic effects, and we investigated their mechanisms related to NF‐κB and ER‐stress pathways. Methods Luciferase, nitric oxide, and cytokine assays using HT‐29 or RAW264.7 cells were conducted. Mouse colitis was induced using dextran sulfate sodium and confirmed by disease activity index and histology. Macrophages and T‐cell subsets in isolated peritoneal cavity cells and splenocytes were analyzed by flow cytometry. Gene and cytokine expression profiles were determined using reverse‐transcription polymerase chain reaction. Results Lactobacillus acidophilus (LA1) and Pediococcus pentosaceus inhibited nitric oxide production in RAW264.7 cells, but only LA1 inhibited Tnfa and induced Il10 expression. LA1 increased the lifespan of dextran sulfate sodium‐treated mice and attenuated the severity of colitis by inducing M2 macrophages in peritoneal cavity cells and Th2 and Treg cells in splenocytes. The restoration of goblet cells in the colon was accompanied by the induction of Il10 expression and the suppression of pro‐inflammatory cytokines. Additionally, we found that LA1 exerts an anti‐colitic effect by improving ER stress in HT‐29 cells as well as in vivo. Conclusions We showed that LA1 significantly interferes with ER stress and suppresses NF‐κB activation. Our findings suggest that LA1 can be used as a potent immunomodulator in IBD treatment, and the regulation of ER stress may have significant implications in treating IBD.