Polysaccharides, one of the active ingredients in herbal medicine, are proved to enhance innate immunity against infections. The aim of this study is to explore the immunoregulatory ability of polysaccharide fraction from R. minima root in vitro and in vivo. Polysaccharide fraction PRM3 from R. minima root were obtained by DEAE-52 cellulose and Sephadex G-150 chromatographic column. PRM3 was nontoxic to macrophages at  8 mg/mL and could strongly enhance the phagocytic ability of macrophages. PRM3 remarkably promoted the release of NO and the secretion of cytokines (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) in macrophages. Simultaneously, PRM3 potently activated NF-κB signaling pathway via Toll-like receptor 4. In addition, PRM3 obviously increased the levels of serum cytokines, markedly up-regulated the percentages of CD3+ and CD4+ T lymphocytes and the CD4+/CD8+ ratio of the splenocytes, and effectively attenuate cyclophosphamide induced immunosuppression in mice. In summary, PRM3 profoundly enhanced the immune function in vitro and in vivo and is a promising candidate of immunopotentiator which could be applied in functional foods or drugs.