Pythiosis is a life-threatening infectious disease caused by the pathogenic oomycete Pythium insidiosum. This study is the first to evaluate the P. insidiosum glucan content and its biological activities. The enzymatic quantification of the glucans in P. insidiosum mycelia showed that the beta-glucan content was 18.99%+/- 3.59. The cell wall polysaccharide extract consisted of similar to 81.7% carbohydrates (exclusively glucose) and similar to 18.3% residual amino acids and peptides. The results from monosaccharide composition, methylation and 1D/2D NMR spectroscopy analyses indicated the presence of a highly branched (1,3)(1,6)-beta-D-glucan, with (1,6)-beta-D-glucopyranosil side-branching unit on average every 1-2 repeat units. In vitro, the beta-D-glucan extract could significantly promote spleen lymphocyte proliferation in human, equine and mouse cell cultures. BALB/c mice that were subcutaneously pre-immunized with three doses of 0.5, 2.5 and 5.0 mg of beta-glucan/mouse, showed a significant increase in IL-2, IL-6, IL-10, TNF-alpha and IL-17A production compared to non-immunized mice. These results suggested that beta-D-glucan extract induces significant and specific Th17 cellular immune response and provided the theoretical basis for further experiments. (C) 2016 Elsevier Ltd. All rights reserved.