AbstractStachyose (STA) is extracted from sugar beet and is used as a functional food. In this study, the effects of STA on immunosuppressed mice with intestinal mucosal damage induced by cyclophosphamide (CTX) are investigated. STA treatment can improve the amount of the leukocyte in the blood, the bone marrow cell density, and natural killer (NK) cell activity in the immunosuppressed mice. Furthermore, in immunosuppressed mice, the balance of intestinal bacteria is broken and intestinal mucosal integrity is disrupted. After treatment with STA, the amount of Enterococcus (6.504 ± 0.70) decreases and Bifidobacterium (7.207 ± 0.14) in cecum increases significantly (p < 0.05). Compared with CTX group, the ratio of villous height and crypt depth (V/C) in ileum increases from 2.525 ± 0.10 to 4.891 ± 0.16 (p < 0.001), and in jejunum increases from 2.719 ± 0.03 to 5.827 ± 0.19 (p < 0.001). The expression level of IL‐2 and IL‐1β gene in the spleen, and IL‐1β and tumor necrosis factor alpha gene in the ileum in STA group significantly increases (p < 0.001) compared with the CTX group. In conclusion, STA is a potential prebiotic which exerts indirect and direct effects on the immune system and can counteract the side effects of CTX in the immunosuppressed mice.