Rhynchosia minima root, a renowned traditional Chinese medicinal herb, had shown multiple therapeutic activities against boils, upper respiratory infections, and joint pains. As one of the active ingredients in R. minima root, polysaccharides were crucial for revealing its biological activities, such as antioxidant, anticancer, and immunoregulatory properties. So we firstly isolated polysaccharides from R. minima root (PRM) using ultrasound-assisted technology and purified them using DEAE-52 cellulose and sephadex G-150 column chromatography. Moreover, we elucidated the structural features of these purified polysaccharide fractions with chemical and physical methods, as well as their antioxidant, anticancer and immunoregulatory activities. We finally determined the anti-inflammatory activity of ethanol fraction from R. minima root. The results showed that the optimal extraction parameters were as follows: ultrasound exposure time, 21 min; ratio of water to material, 46 mL/g; and ultrasound extraction temperature, 63 °C. Under these conditions, the maximum yield of PRM was 16.95% ± 0.07%. Three acidic polysaccharide fractions, named PRM1, PRM3 and PRM5, were purified. The uronic acid contents of PRM1, PRM3 and PRM5 were 30.7, 12.7 and 47.7%, respectively. PRM1 (143.2 kDa), PRM3 (105.3 kDa) and PRM5 (162.1 kDa) were heteropolysaccharides because they were composed of arabinose, mannose, glucose and galactose. Their enthalpy values were 201.0, 111.0 and 206.8 J/g, respectively. PRM3 and PRM5 exhibited remarkable DPPH radical scavenging activities and reducing power in vitro. PRM3 showed strong inhibitory activities on the growth of human breast cancer cells MCF-7 in vitro. PRM3 and PRM1 exhibited strong in vitro inhibitory activity against human lung cancer cells A549 and human liver cancer cells HepG2 in a dose-dependent manner. PRM3 displayed an excellent α-glucosidase inhibitory activity with an IC50 value of 8.85 mg/mL, which was similar to that of acarbose. Referred to above results, we elucidated the structural features of PRM3 detailedly. The repeating units of PRM3 were mainly composed of (1→3,4)-α-Arap, (1→4,6)-β-Galp, (1→2,4)-β-Rhap, (1→3,4)-β-GalpA, T-α-Araf, and T-α-Galp residues. The triple helix stereo-configuration also existed in PRM3. Importantly, the immunoregulatory ability of PRM3 was evaluated in vitro and in vivo. PRM3 remarkably enhanced the phagocytic ability of macrophages and promoted the release of NO and the secretion of cytokines (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6; and monocyte chemoattractant protein-1, MCP-1) from macrophages. Simultaneously, PRM3 potently activated NF-κB signaling pathway via Toll-like receptor 4 (TLR4). 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 attenuated cyclophosphamide induced immunosuppression in mice. In addition, ethanol fraction 6 (EEF6) of R. minima root exhibited strong anti-inflammatory ability through decreasing the secretion of NO, IL-6, TNF-α, and MCP-1, inhibiting the translocation of p65 from cytoplasm to nucleus, and suppressing the phosphorylation of ERK, JNK, and p38. Additionally, five flavonoids compounds in EEF6 were identified. This study indicated that active fractions from R. minima root, polysaccharides and EEF6, were the promising candidates to regulate the inflammatory level in body.