It has been reported that lentinan, β-1,3; 1,6-glucan derived from Lentinula edodes, suppresses intestinal inflammation and ameliorates symptoms of colitis. However, the mechanism of intestinal anti-inflammatory activity of lentinan and how it is recognized by intestinal epithelial cells remains largely unclear. The receptor for lentinan on intestinal epithelial cells was identified using an in vitro gut inflammation model consisting of Caco-2 and RAW264.7 cells. Colitis was induced in 7 to 8 week-old wild-type (WT) or knockout (KO) mice by the free intake of water containing 2% dextran sulfate sodium (DSS) for 7 days. Lentinan was administered daily via intragastric administration. Tumor necrosis factor receptor 1 (TNFR1)-GFP complex was constructed to monitor its movement in Caco-2 cells using confocal and total internal fluorescence microscopy. The results indicated that lentinan suppressed DSS-induced body weight loss, shortening of colon length, histological score, and inflammatory cytokine mRNA expression in the inflamed tissues of WT mice, whereas these suppressive effects of lentinan were not observed in Dectin-1 KO mice. Furthermore, lentinan reduced TNFR1 expression in intestinal epithelial cells of WT mice but not those from Dectin-1 KO mice. Using TNFR1-GFP constructs, it was confirmed that lentinan reduced TNFR1 expression on Caco-2 cell membranes through Dectin-1 ligation. Our study revealed that lentinan suppressed intestinal inflammation by Dectin-1-mediated regulation of TNFR1 transfer to the surface of intestinal epithelial cells.