The aim of this study was to evaluate inhibitory effect of glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on allergic responses. We evaluated the activities of mitogen-activated protein kinase (MAPK), transcriptional factor, and production of immunoglobulin (Ig)E and interleukin (IL)-4 in RBL-2H3 cells and BALB/c mice. Our results showed that CTB glycoprotein inhibited the production of IgE and IL-4 in serum from ovalbumin (OVA)-treated BALB/c mice. We also found that CTB glycoprotein inhibited the phosphorylation of p38 MAPK, and transcriptional activation of nuclear factor (NF)-κB in RBL-2H3 cells. The activation of NF-κB was effectively blocked by treatment with p38 MAPK inhibitor (SKF86002). The results from these experiments indicate that the CTB glycoprotein inhibits release of β-hexosaminidase, and production of IgE and IL-4 via down regulation of MAPK/ NF-κB on the stage of mast cell degranulation. In conclusion, we suggest that the CTB glycoprotein might be a potent preventive agent in allergic responses.