The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264·7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), and Interleukin-1β (IL1-β)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from β-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-α showed a direct correlation between protein concentration and TNF-α release. The fraction contained four novel peptides, three derived from the hydrolysis of β-casein and one from the hydrolysis of α-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.