Macrophages play a pivotal role in the innate and adaptive immune systems. This study investigated the immuno-modulatory activities of polysaccharides separated from Chrysanthemum zawadskii var. latilobum (CZPS) in macrophages. Polysaccharides from Chrysanthemum zawadskii var. latilobum were extracted by the ethanol precipitation method. RAW 264.7 mouse macrophage cell line was treated with CZPS (4 to 128 μg/mL), and there was no cytotoxicity at a dose below 32 μg/mL. The levels of nitric oxide (NO) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6, IL-1β) production in the CZPS treated group (32 μg/mL) were 6.5±0.12 μM (NO), 1252.8±79.85 (TNF-α), 305.4±29.41 (IL-6), and 683.3±59.71 (IL-1β), respectively, and they were significantly increased when compared to the control group; 2.2±0.03 μM (NO), 452.3±38.34 (TNF-α), 31.7±5.75 (IL-6), and 184.1±11.52 (IL-1β). Additionally, protein expression of inducible nitric oxide synthase (iNOS) and phosphorylation of MAPKs and NF-κB expression were significantly increased upon CZPS treatment. Therefore, these results indicated that polysaccharides separated from Chrysanthemum zawadskii var. latilobum (CZPS) may have a potential immunomodulatory activity in macrophages through MAPKs and NF-κB signaling, and this information is useful for the development of immune enhancing adjuvant materials using a natural ingredient.