To utilize pepper (Piper nigrum) as an immunostimulatory agent, we isolated macrophage stimulating polysaccharides from pepper and investigated their macrophage activating activities. Hot-water extracts (HW) of black pepper (BP) and white pepper (WP) were prepared, and their macrophage stimulating activities were evaluated using RAW 264.7 cells. BP-HW significantly promoted the secretion of macrophage stimulating factors such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-12 compared to WP-HW. When BP and WP-HW were fractionated into crude polysaccharides (CP) and low molecules (LM) by ethanol precipitation, BP-CP demonstrated significantly more potent activity. Furthermore, BP-CP not only induced mRNA gene expression of macrophage activation factors, but also promoted nuclear localization of p65 and c-Jun. In addition, component sugar analysis revealed that glucan-type polysaccharides in BP-CP played a crucial role in macrophage activation. Taken together, these findings suggest that black pepper has industrial applicability not only as a spice, but also as an immunostimulatory functional material.