Adenosine is an endogenous signaling molecule upregulated during inflammatory conditions. Acting through the A2b receptor (A2bR), the predominant adenosine receptor in human colonic epithelia, adenosine has been directly implicated in immune and inflammatory responses in the intestine. Little is known about expression and regulation of A2bR during inflammation. Tumor necrosis factor alpha (TNF-alpha) is highly upregulated during chronic and acute inflammatory diseases. This study examined the expression of A2bR during colitis and studied effects of TNF-alpha on A2bR expression, signaling and function. Results demonstrated that A2bR expression increases during active colitis. TNF-alpha pretreatment of intestinal epithelial cells increased A2bR messenger RNA and protein expression. TNF-alpha significantly increased adenosine-induced membrane recruitment of A2bR and cyclic adenosine monophosphate downstream signaling. Further, TNF-alpha potentiated adenosine-induced shortcircuit current and fibronectin secretion. In conclusion, we demonstrated that TNF-alpha is an important regulator of A2bR, and during inflammation, upregulation of TNF-alpha may potentiate adenosine-mediated responses.