Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1 (RAMP1). We used RNA interference (RNAi) to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days and the effect of knockdown spread to approximately 2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone, but slow wave contractions of varying amplitude persisted. In TNBS-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. The pro-inflammatory cytokines TNF- and IL-6 levels were markedly upregulated by TNBS-treatment. TNF- mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus, this study demonstrates that CLR is a functional receptor for CGRP.