A water-soluble polysaccharide (BP-1) was extracted and purified from highland barley (Hordeum vulgare L.) and its average molecular weight was about 6.7 × 104 Da. In this study, the immunomodulatory activity of BP-1 on the immunosuppressive BALB/c mice model and its molecular mechanism were elucidated. It was found that the weight indexes of spleen and thymus were significantly increased by BP-1 (80 mg kg-1 and 160 mg kg-1) treatment in the immunosuppressive mice model. The results showed that BP-1 (80 mg kg-1 and 160 mg kg-1) could significantly increase the number of bone marrow cells (BMC) and peripheral blood white blood cells (WBC) in the immunosuppressive mice model. In addition, the result further confirmed that BP-1 could increase the serum levels of IL-2, TNF-α and IFN-γ, so as to improve the immune function of immunosuppressive mice. The results showed that BP-1 (80 mg kg-1 and 160 mg kg-1) could promote the proliferation of spleen cells and the natural killer (NK) cell activity in vivo. The quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA results revealed that BP-1 (80 mg kg-1 and 160 mg kg-1) could enhance the production of IL-2, TNF-α, IFN-γ, IgG and IgM in the spleen of immunosuppressive mice. The HE (hematoxylin and eosin) stained histopathological images showed that BP-1 (80 mg kg-1 and 160 mg kg-1) could repair the damage induced by CTX in the spleen cells of immunosuppressive mice. The result of macrophages showed that BP-1 (80 mg kg-1 and 160 mg kg-1) could promote the proliferation and phagocytosis activity of macrophages in immunosuppressive mice. Furthermore, BP-1 could activate macrophages by the TLR-4, TRAF6, TAK1 and nuclear factor κB (NF-κB) p65 pathways in vivo. These results suggested that BP-1 has a remarkable immunomodulatory activity on the immunosuppressive mice model.