This study evaluated effect of triterpenoids from edible mushroom Poria cocos on intestinal epithelium integrity and revealed the transcriptional regulatory pathways that underpin restorative mechanisms in the gut. Based on computational docking studies, transcriptional activation experiments, and glucocorticoid receptor (GR) protein immunofluorescence localization assays in cultured cells, 16α-hydroxytrametenolic acid (HTA) was discovered as a novel GR agonist in this study. HTA ameliorates TNF-α induced Caco-2 monolayer intestinal epithelial barrier damage and suppressed activation of PI3K and Akt which attenuated downstream IκB and NF-κB phosphorylation through GR activation. Moreover, HTA prevented NF-κB translocation into the nucleus and binding to its cis-element and suppressed LPS-induced downstream NO production, pro-inflammatory cytokines at both protein and mRNA expression levels. In conclusion, HTA from P. cocos improves intestinal barrier function through a GR mediated PI3K/Akt/NF-κB signaling pathway and may be potentially exploited as a supportive dietary therapeutic strategy for restoring gut health.