Background: Polysaccharide has played the part of great role in pharmacology and physiology. Materials and Methods: In this study, the polysaccharides (CIS-A) from Catathelasma imperiale (Fr.) Sing, were isolated and purified by hot water extraction technology and column chromatography, respectively. Chemical methods, infrared spectrum, high-performance gel-permeation chromatography, high-performance liquid chromatography, gas chromatography–mass spectrometry,1H nuclear magnetic resonance spectroscopy (NMR),13C NMR, and two-dimensional NMR were used to characterize the polysaccharides of CIS-A. The anticancer and immunomodulatory ability of the polysaccharides (CIS-A) from the fruiting body of C. imperiale (Fr.) Sing was also investigated. Results: The structural feature analysis showed the polysaccharide (CIS-A) which had a molecular weight of 50486 Da was mainly composed of α-D-glucose pyranose (α-D-Glcp) and β-L-fucose pyranose (β-L-Fucp). It had a backbone of three 1, 3-linked α-D-Glcp. There is a branch at the C2 of the polysaccharide backbone. The branches were mainly composed of two 2, 3-linked β-L-Fucp residue. Antitumor activity results showed that CIS-A could inhibit the growth of S180 tumor and promote the apoptosis of L929 cells. Immunoregulatory activity results showed that CIS-A could promote the proliferation of T-cells and promote B-cells by affecting G0/G1 phase, S phase, and G2/M phase. It also could promote the proliferation and phagocytosis of macrophages and induce cytokine release. Conclusion: Polysaccharide CIS-A can be used as a candidate drug for antitumor and immunomodulator.