Mushroom polysaccharides have been reported to possess significant biological activities. However, their molecular mechanisms are not fully elucidated. In this study, the immunostimulating activity of a newly purified heteropolysaccharide L2 from Lentinula edodes is evaluated in Caco-2 cells and a Caco-2/RAW264.7 co-culture system, as well as in mice. Subsequently, the customized RT-PCR array containing 112 genes is employed to investigate the effects of L2 on gene expressions in the small intestine, cecum and colon. The results show that L2 significantly enhances immune responses by differentially affecting the gene expressions of small intestine, cecum and colon, in which 55, 26 and 25 genes are markedly changed, respectively. In particular, 3 core regulation networks are identified for various parts of gut. These data demonstrate the potential of L2 as a potent immune stimulator and for the first time provide a detailed landscape of tissue-specific gene expressions and core regulation networks in response to L. edodes-derived heteropolysaccharide treatment.