Selenium-enriched exopolysaccharides (Se-EPS) produced by Lactococcus lactis subsp. lactis can be used as a safe and effective selenium (Se) supplement. Analysis of the Se content and monosaccharide components of Se-EPS demonstrated that it consisted of mannose, fucose, ribose, glucose, galactose, and arabinose with a molar ratio of 5.48 : 0.39 : 9.77 : 4.03 : 1.00 : 1.92, and an Se content of 183.263 μg g(-1). The differing effects of Se-EPS and EPS on calcium channels and certain key secondary messengers in spleen lymphocytes were examined and compared. Results showed that low-dose Se-EPS, but not EPS, increased the intracellular calcium ([Ca(2+)]i) levels of mouse spleen lymphocytes. Se-EPS also increased the expression and phosphorylation of Ca(2+)-calmodulin-dependent kinase (CaMK) II in lymphocytes. In addition, increased intracellular Ca(2+) levels were also found in the cells with blocked Ca(2+) channels. We speculated that Se-EPS enhanced the activation and proliferation of lymphocytes through calcium signaling by drawing from extracellular and intracellular stores. Low-dose Se-EPS also enhanced NO production, cAMP levels and PKA activity. We speculated that low-dose Se-EPS may activate certain pathways, including the calcium channel, NO, cAMP, and PKA related pathways.