Xylooligosaccharides (XOs) are considered as food ingredients, and exhibit the prebiotic effects related to gut modulation. However, no related research is available to explain their immunomodulatory effects. This report elucidated their immunomodulatory effects through the variations of proinflammatory mediators in vitro. We found that XOs (0.1 − 100 μg/mL) induced tumor necrosis factor alpha (TNF-α), IL-1β, IL-6 and nitric oxide (NO) production in un-stimulated macrophages, RAW264.7 cells. Furthermore, pre- and post-treated XOs (0.1−100 μg/mL) dose-dependently suppressed TNF-α, IL-1β, IL-6 and NO production and induced IL-10 production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells without exerting cytotoxicity. Of note is that prostaglandin E₂ (PGE₂) production didn’t change significantly through the XOs treatment. These data demonstrate that XOs potently down-regulates the LPS-induced inflammatory response. The in vitro assessment of inflammatory mediators should be useful in further characterization of the effects of XOs on immunomodulation.