Gut microbiota (GM) contributes to the production of immune-regulatory molecules and cytokines. However, our understanding regarding intricate relationship between Lactobacillus plantarum and GM on regulation of immune function remained limited. To investigate the effect of Lactobacillus plantarum on an immunosuppressed mouse model, we employed cyclophosphamide treatment and conducted various analysis including H&E (hematoxylin-eosin staining), immunohistochemistry, 16S rRNA gene sequencing, and RT-PCR. Our results demonstrated that the administration of Lactobacillus plantarum had significant immunoenhancing effects in the immune-suppressed mice, as evidenced by the restoration of functional expression of specific immune markers in the spleen and an increase in the number of goblet cells in intestine (P < 0.05). Microbial taxonomic analysis revealed alterations in the gut microbiota composition, characterized by a decrease in the richness of Firmicutes and an increase in the proportion of Verrucomicrobia and Actinobacteria following cyclophosphamide treatment. Furthermore, cyclophosphamide treatment significantly suppressed the mRNA expression of inflammatory cytokines (P < 0.05), which were subsequently restored after administration of Lactobacillus plantarum. These observations provide valuable insights into the complex interplay between probiotics, gut microbiota, and immune system functioning.