Exopolysaccharides (EPSs) are a metabolite of probiotics which have gained wide interest recently, but little is known about their function. EPS was isolated from Bifidobacterium longum BCRC 14634 and sterilized by 0.22μm filter. The proliferation of J77A.1 macrophages and their secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) was elevated after treatment with heat-killed B. longum or 5μg/mL EPS. The endotoxin, lipopolysaccharides (LPS), a potent inducer of pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), significantly suppressed the growth of J77A.1 cells, and induced the secretion of TNF-α from J774A.1 cells. Furthermore, 24h pretreatment with 5μg/ml EPS suppressed 100ng/ml LPS-induced cell growth inhibition and release of TNF-α from J774A.1 cells. Additional experiments showed that 80μg/mL EPS had antimicrobial activity against 7 species of food-spoilage and infection bacteria. Our results suggest that EPS from B. longum might be useful as a mild immune modulator for macrophages, contributing to the capacity of B. longum to fight against gastrointestinal infections, and even some food-spoilage microbe.