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Lactobacillus rhamnosus (LBS) is a well-documented probiotic strain in oncology and has a pivotal role in clinical applications. Here, we have investigated the protective effect of Lactobacillus rhamnosus on intestinal mucositis induced by cisplatin (CP) and explored the underlying mechanisms targeting inflammatory proteins, as well as the histological changes in the intestinal tissue of mice, in addition, the bacterial strains that may be related to the health-enhancing properties. BALB/c mice were pre-treated with or without LBS via oral gavage, followed by mucositis induction with cisplatin. Our results revealed that the LBS-treated groups significantly attenuated proinflammatory cytokine levels (IL-1β, IL-6, and TNF-α) compared to the CP group. Furthermore, LBS mitigated the damaged tight junction integrity caused by CP via up-regulating the levels of claudin, occludin, ZO-1, and mucin-2 protein (MUC-2). Finally, the 16S rRNA fecal microbiome genomic analysis showed that LBS administration enhanced the growth of beneficial bacteria, i.e., Firmicutes and Lachnospiraceae, while the relative abundance of the opportunistic bacteria Bacteroides and Proteobacteria decreased. Collectively, LBS was found to beneficially modulate microbial composition structure and functions and enrich the ecological diversity in the gut.