beta-1,4-Mannobiose (MNB) supplementation has been shown to prevent Salmonella Enteritidis infection in broilers by improving Salmonella Enteritidis clearance and increasing IgA production. This study examined in detail the gut immunomodulatory activity of MNB using microarray and real-time quantitative PCR analysis. One-day-old chicks were orally administered 0.1% (wt/wt) MNB 3 times a week for 28 d. Control birds received vehicle alone. Body weights and fecal IgA levels were monitored weekly. On d 28, spleen and bursa of Fabricius were removed and weights were recorded; samples of ileum, jejunum, cecum, spleen, thymus, and bursa of Fabricius were collected for histological examination; and ileum samples were collected for RNA extraction. No significant difference in BW or organ weights was observed between MNB-treated and untreated control birds, and no histological abnormalities were observed in any of the tissues examined. The MNB-treated chickens had significantly higher levels of fecal IgA over all 4 wk when compared with control birds. Microarray and reverse transcription PCR analysis revealed the upregulation of several genes involved in immune responses, including those involved in antigen recognition, processing and presentation (MHC class I and II), interferon-related genes, and genes involved in host defense. These results provide insight into the mechanism of action of dietary MNB in the intestine and confirm that MNB acts as a potent immune-modulating agent, exerting combined effects on the intestinal immune system.