Display omitted •A novel exopolysaccharide (R-5-EPS) was obtained from Lactobacillus helveticus LZ-R-5.•R-5-EPS was a linear hetero-glucogalactan with average Mw of 5.41 × 105 Da.•Structure of R-5-EPS was composed of →3)-β-D-Glcp-(1→ and →6)-β-D-Galp-(1→ residues.•R-5-EPS showed potent macrophage immunomodulatory activity in vitro. Exopolysaccharide (R-5-EPS) was isolated from the fermented milk of Lactobacillus helveticus LZ-R-5 and purified by DEAE-52 cellulose anion-exchange column, and characterization of the structure was conducted. Results showed that R-5-EPS was a heteropolysaccharide containing linear repeating units of →6)-β-D-Galp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ with an average Mw of 5.41 × 105 Da. Furthermore, at a cellular level, R-5-EPS showed immunostimulatory activity due to its strong effect on increasing proliferation of RAW264.7 macrophages and enhancing phagocytosis, acid phosphatase activity, nitric oxide production and cytokines production in macrophages. These results suggest that R-5-EPS have a potent immunostimulatory activity and may be explored as a potential immunomodulatory agent.