AimsThe aim of this study was to investigate the immunostimulatory effects of an exopolysaccharide-enriched fraction obtained from Bacillus subtilis J92 (B-EPS). Methods and ResultsTo determine the immunostimulatory activities of B-EPS, we used IFN--primed RAW 2647 macrophages and CD3/CD28-stimulated splenocytes. Increases in the levels of NO and many cytokines, such as, TNF-, IL-6, and IL-1, were observed in IFN--primed RAW 2647 macrophages by Griess reaction and ELISAs respectively. Using Western blotting and qRT-PCR, we found that B-EPS increased the protein and mRNA expressions of iNOS and the mRNA expressions of TNF-, IL-6, and IL-1. A reporter gene assay and EMSA revealed that B-EPS up-regulated the transcriptional activity of NF-B by increasing its DNA binding and nuclear translocation. Pretreatment with NF-B inhibitors, that is, BAY11-7082 and PDTC, decreased NO production in IFN--primed RAW 2647 macrophages by B-EPS. Furthermore, B-EPS increased the proliferation of and cytokine (IL-2 and IFN-) production by CD3/CD28-stimulated splenocytes. In a cyclophosphamide-induced immunosuppressed mouse model, B-EPS (5, 15 or 45mgkg(-1), p.o.) restored thymus and spleen indices. B-EPS also inhibited cyclophosphamide-induced reductions in neutrophil and lymphocyte numbers. ConclusionsB-EPS improves immune function by regulating immunological parameters in IFN--primed macrophages, CD3/CD28-stimulated splenocytes, and in cyclophosphamide-induced immunosuppressed mice. Significance and Impact of the StudyThis study suggests that the exopolysaccharides secreted by B.subtilis J92 could be used as immune stimulants.