Barley is commonly used in many food and health products. We have previously demonstrated the macrophage-stimulating properties of polysaccharides derived from fermented barley. In this study, three polysaccharide fractions (BF-I-III) were purified from fermented barley and their monosaccharide composition was analyzed. Their immune-stimulatory activities and intracellular signaling pathways were also studied in RAW264.7 cells. Among the three fractions, BF-I exhibited enhanced macrophage activation properties, such as inducing the production of IL-6, IL-12, and TNF-α. However, BF-II and BF-III showed moderate effects on RAW 264.7 cells. BF-I treatment led to the phosphorylation of MAPKs, NF-κB, and c-Jun (major component of AP-1 transcription factor) and induced the nuclear translocation of p65 in RAW264.7 cells. In addition, experiments with neutralizing antibodies showed that Dectin-1, toll-like receptor (TLR) 4, scavenge receptor (SR), and CD14 were mainly involved in the stimulation of nitric oxide (NO) production by BF-I which was suppressed by the inhibition of JNK phosphorylation. These findings suggest that BF-I, isolated from fermented barley, has an immune potentiation activity on macrophages, where it activates the JNK signaling pathway via several macrophage receptors including dectin-1, TLR4, SR, and CD14.