Receptor activator of NF-κB (RANK) and its ligand (RANKL) are important members of the TNF receptor (TNFR) and TNF superfamilies, respectively. RANK is expressed on osteoclasts, T-lymphocytes, and dendritic cells, and its ligation with RANKL leads to cellular activation. However, another member of the TNFR family, osteoprotegerin (OPG), acts as a decoy receptor, binding to RANKL and preventing its interaction with RANK. Furthermore, OPG also binds TNF-related apoptosis-inducing ligand (TRAIL), an important regulator of cell survival. OPG is therefore an important regulator of bone metabolism and immune responses. Although intestinal epithelial cells (IEC) express some members of the TNF/TNFR superfamilies, the roles of OPG and RANKL in the intestinal mucosa has not been investigated. Here, we report that various human IEC lines constitutively express OPG mRNA and protein as well as mRNA for RANKL. Furthermore, human colonic epithelium constitutively expressed OPG, and this expression was increased in inflamed tissue. All of the IEC lines tested released OPG into the culture supernatant under standard culture conditions. Whereas TNF-α increased OPG protein secretion by HT29 cells, the cytokines IL-1β and IFN-γ had little, if any, effect. Furthermore, the culture supernatant from untreated HT29 cells abrogated TRAIL-induced inhibition of Jurkat T-cell proliferation and inhibited osteoclast activity in an in vitro model of bone resorption. Taken together, our data indicate that OPG is constitutively produced by IEC, could be upregulated by TNF-α, and is biologically active. Thus IEC-derived OPG may represent an important mucosal immunoregulatory factor and may be involved in bone physiology.