Delayed colonic emptying leading to constipation is a significant health concern. We investigated the role of adenosine 2B receptor (A_2BAR) in modulating distal colonic motility using wild-type and A_2BAR-knockout (A_2BAR^-/-) mice. Colon motility was assessed using stool characteristics and colonic transit. Distal colonic ganglia, isolated by laser capture microdissection, were tested for A_2BAR expression by RT-PCR. The distal colon contraction and relaxation responses were assessed by electrical field stimulation (EFS) in presence of A_2BAR agonists, antagonists or inhibitors of nitric oxide (NO) and guanylate cyclase. Nitrite levels were measured in enteric neuronal cultures exposed to A_2BAR agonists/antagonists. A_2BAR^-/- mice display increased stool retention, decreased stool frequency, delayed colonic emptying, and decreased circular muscle relaxation. RT-PCR identified A_2BAR expression in distal colonic ganglia. EFS studies revealed that enteric neuronal A_2BAR is essential for distal colonic relaxation, and A_2BAR antagonists can inhibit relaxation. Enteric neurons stimulated with A_2BAR agonists produced more nitrite than cultures treated with antagonists. We demonstrate an essential role of A_2BAR in regulating distal colon relaxation, as A_2BAR activation is linked to NO signaling. Hence targeting the colonic A_2BAR could represent a novel therapeutic strategy to treat constipation.—Chandrasekharan, B. P., Kolachala, V. L., Dalmasso, G., Merlin, D., Ravid, K., Sitaraman, S. V., Srinivasan, S. Adenosine 2B receptors (A_2BAR) on enteric neurons regulate murine distal colonic motility.