Helicteres angustifolia L. (H. angustifolia) has been widely used as a remedy against various types of illness relating to immune response, such as inflammations and fever. In order to characterize the structure and identify the immunomodulatory activity of polysaccharide from H. angustifolia, a polysaccharide fraction (SPF3-1) was purified from H. angustifolia by using DEAE Sepharose Fast Flow and Sephacry S-400 chromatography, successively. Physicochemical analysis demonstrated that SPF3-1 is an acidic heteropolysaccharide with a molecular weight of about 13.36 kDa; in vitro immunomodulatory assay reflects that SPF3-1 could significantly (p <  0.05) enhance the proliferation of macrophages, stimulate the macrophages phagocytic capacity, as well as induce NO and immunomodulatory cytokines generation. All the results suggest that SPF3-1 from H. angustifolia possesses potent immunomodulatory activity and could be further developed as new products for medicines or functional foods.