In this study, the strain Lactiplantibacillus plantarum DLPT4 was investigated for the immunostimulatory activity in cyclophosphamide (CTX)-induced immunosuppressed BALB/c mice. L. plantarum DLPT4 was administered to BALB/c mice by oral gavage for 30 days, and CTX was injected intraperitoneally from 25th to 27th days. Intraperitoneally CTX injection caused the damage to the thymic cortex and intestines, and the immune dysfunction of the BALB/c mice. While L. plantarum DLPT4 oral administration exerted immunoregulating effects evidenced by increasing the serum immunoglobulin A (IgA), IgG and IgM levels, and reducing the expression of pro-inflammatory factors (IL-6, IL-1β and TNF-α) of the BALB/c mice with intraperitoneally CTX injection. The results of the BALB/c mice feces metagenome-sequencing showed that L. plantarum DLPT4 oral administration can regulate the intestinal microbial community by changing the ratio of Lactiplantibacillus and Bifidobacterium. Meanwhile, the abundance of carbohydrate enzyme (CAZyme), immune diseases metabolic pathways and p38 and AP-1 MAPK signaling pathways were enriched in the BALB/c mice with L. plantarum DLPT4 oral administration. In conclusion, L. plantarum DLPT4 administration ameliorated CTX-induced immunosuppression in BALB/c mice by regulating gut microbiota, and further influencing the abundance of carbohydrate esterase in the intestinal flora and increasing the immune metabolic activity. L. plantarum DLPT4 could be used as promising probiotics to regulate immune response.