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A previous study demonstrated that Lactobacillus(L.) plantarumNCU1125, derived from fermented Nanfeng mandarin, survived in vitrosimulated gastrointestinal digestion, suggesting potential probiotic properties. The current study aimed to investigate the efficacy of L. plantarumNCU1125 on mice of cyclophosphamide-induced intestinal barrier (including the physical, the immunologic and the microbial barriers) injury. The results showed that L. plantarumNCU1125 could repair the intestinal physical barrier through upregulating the expressions of Muc2 and tight junctions (ZO-1, occluding and claudin-1), strengthen the intestinal immunologic barrier through boosting the number of CD4+T cells (64.34%), advancing the secretion of certain cytokines (IL-6 (2.77 times), IL-10 (1.55 times), IL-17 (1.19 times), TGF-β3 (2.42 times), IgA (2.42 times) and sIgA (1.42 times)), inducing Th (RORγt (31.06%) and Foxp3 (57.82%)) and B cells differentiation (Blimp-1 (21.10%), IRF-4 (41.27%) and XBP-1 (37.18%)), and stimulating sIgA formation (joining chain (98.68%), IgA α-chain (71.61%) and pIgR (36.75%)), and reshape the microbial barrier through modulating gut microbiota and metabolism. In addition, activation of the TLR4/MyD88/NF-κB pathway by L. plantarumNCU1125 might mediate expressions of gene products involved in the intestinal physical barrier, Th cell differentiation and sIgA secretion. Moreover, L. plantarumNCU1125 was found to control intestinal inflammation by correcting the imbalance of Th17/Treg. All findings indicated that L. plantarumNCU1125 could attenuate the intestinal barrier injury caused by cyclophosphamide in mice and lay a theoretical foundation for its applications in the intestinal regulation.