We here extracted a polysaccharide fragment from Gastrodia elata, characterized by a main chain connected via (1 → 4)-α-D-Glcp bonds, with terminal α-D-Glcp-(1→) linked to the main chain through O-6 of (1 → 4,6)-α-D-Glcp and O-3 of (1 → 3,4)-α-D-Glcp (SRGP). Both in vitro and in vivo experiments demonstrated that SRGP activates the TLR4/NF-κB signaling pathway, exerting immunomodulatory effects and alleviating cyclophosphamide (CTX)-induced intestinal mucosal damage in mice. High-throughput 16S rRNA sequencing further revealed that SRGP restores gut microbiota composition and enhances the abundance of specific bacterial populations. Additionally, SRGP improves CTX-induced intestinal mucosal damage by upregulating tight junction proteins, mitigating gut microbiota dysbiosis, and regulating both the overall microbial community and the levels of specific bacteria.