Much research suggests that a dietary supplement of Chlorella pyrenoidosa may be helpful to human health, but the molecular mechanism involved remains unclear. The aim of this research was to investigate the effects of certain hot-water-soluble polysaccharides from Chlorella pyrenoidosa (CWSP) on cytokine production, human leukocyte antigen (HLA) expression, and costimulatory molecule expression in macrophages. We demonstrated that CWSP induced IL-1beta secretion in macrophages via Toll-like receptor 4 (TLR4) mediated protein kinase signaling pathways. In addition, CWSP also stimulated the cell surface expression of HLA-DA, -DB, and -DC, and HLA-DR, -DP, and -DQ as well as the expression of costimulatory family molecules such as CD80 and CD86 in macrophages. Furthermore, we demonstrated that preinjection of C57BL/6J mice with CWSP increased lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha and IL-1beta secretion into serum in vivo. This outcome was consistent with the corresponding outcome for cells treated with CWSP in vitro. Our current results provide support for the possible use of CWSP as a modulation agent of immune responses in humans and certain animal species. Finally, in using GC-MS to analyze the polysaccharides, we found that the major monosaccharides of CWSP were rhamnose (31.8%), glucose (20.42%), galactose (10.28%), mannose (5.23%), and xylose (1.27%). This study is the first to report the molecular mechanism of immune-modulated signal transduction in vitro from the polysaccharides of Chlorella pyrenoidosa.