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Polysaccharides from Boswellia carterii were isolated using hot water and fractionated on a DEAE Sepharose Fast Flow column to evaluate their structural characteristics and macrophage stimulating capacities. Crude and fractionated polysaccharides (BCF1 and BCF2) were mainly consisted of different amounts of neutral sugars (63.7–83.3%) and uronic acids (6.2–22.3%). The weight average molecular weights (Mw) of polysaccharides greatly varied between 295.5 and 504.7 × 103 g/mol. The BCF2 was a highly active polysaccharide on RAW264.7 murine macrophage cells inducing considerable release of nitric oxide and proinflammatory cytokines including TNF-α, IL-1β and IL-6. The presence of p-NF-κB, p-JNK, p-ERK and p-p38 proteins in the cytoplasm of the stimulated macrophages indicated the activation of cells via NF-κB and MAPKs signaling pathways which was followed after CBF2-macrophage interaction through TLR4 and TLR2 receptors. The most potent macrophage activating polysaccharide, BCF2, contained a backbone of (1 → 6)-β-galactopyranosyl units branched at C-3.