Exopolysaccharide (EPS) from probiotic Enterococcus hirae WEHI01 was isolated and purified by anion exchange chromatography and gel chromatography, the results of which show that the EPS consists of four fractions, namely I01-1, I01-2, I01-3, and I01-4. As the main purification components, I01-2 and I01-4 were preliminarily characterized for their structure and their immunomodulatory activity was explored. The molecular weight of I01-2 was 2.28 × 104 Da, which consists mainly of galactose, and a few other sugars including glucose, arabinose, mannose, xylose, fucose, and rhamnose, while the I01-4 was composed of galactose only and has a molecular weight of 2.59 × 104 Da. Furthermore, the results of an evaluation of immunomodulatory activity revealed that I01-2 and I01-4 could improve the viability of macrophage cells, improve phagocytosis, boost NO generation, and encourage the release of cytokines including TNF-α and IL-6 in RAW 264.7 macrophages. These results imply that I01-2 and I01-4 could improve macrophage-mediated immune responses and might be useful in the production of functional food and medications.