EPS53, a homogeneous exopolysaccharide (EPS) was isolated from Streptococcus thermophiles XJ53 fermented in skimmed milk via anion exchange column chromatography. The relative molecular weight of EPS53 was above 6.7 × 105 g/mol; its repeating structural unit of EPS53 consisted of β-T-Galp, β-1,3-Galf, α-1,3-Glcp and β-1,3,6-Glcp residues in a molar ratio of 1:1:1:1, with β-T-Galp attached to the O-6 position of β-1,3,6-Glcp,identical to the EPS produced from S. thermophilus SFi39. EPS53-D, purified under similar conditions as EPS53 except for the deproteinization of trichloroacetic acid (TCA), had a lower molecular weight but the same repeating structural unit. The effects of EPS53 and EPS53-D on proliferation, phagocytosis and nitric oxide (NO) release of macrophage RAW264.7 were compared. EPS53 exhibited stronger immune activity than EPS53-D, suggesting that the molecular weight might have an important effect on the activity of EPS molecules. Treatment with TCA might affect the activities of native EPSs produced by fermentation.