Aims: To evaluate the immunosuppressive properties of the exopolysaccharide (EPS) from high-EPS producer Lactobacillus rhamnosus RW-9595M on inflammatory cytokines produced by macrophages. Methods and Results: The conditioned media (CM) were produced by macrophages treated with parental Lact. rhamnosus ATCC 9595 and its isogenic variant, the high-EPS producer Lact. rhamnosus RW-9595M, and the levels of TNF-alpha, IL-6, IL-10 and IL-12 were evaluated. Results revealed that CM from parental Lact. rhamnosus induced higher levels of TNF-alpha, IL-6 and IL-12 but inhibited IL-10 production, whereas its mucous variant induced low or no TNF-alpha and IL-6. Addition of purified EPS to macrophages treated with parental Lact. rhamnosus decreased the inflammatory cytokines and inhibited the metabolic activity of lymphocytes. The intermediate polysaccharide chains (16-30 units) produced by time-controlled hydrolysis of EPS increased the IL-10 produced by macrophages. Conclusions: Polysaccharide chains of EPS induced immunosuppression by the production of macrophagic anti-inflammatory IL-10. Significance and impact of the Study: These results indicate that the EPS from Lact. rhamnosus RW-9595M may be useful as a new immunosuppressive product in dairy food.