Background This study attempted to provide the effects and mechanisms of two cannabinoids, O‐1602 and cannabidiol (CBD), on colonic motility of 2,4,6‐trinitro‐benzene sulfonic acid (TNBS) colitis. Methods TNBS was used to induce the model of motility disorder. G protein‐coupled receptor 55 (GPR55) expression was detected using real‐time PCR and immunohistochemistry in colon. Pro‐inflammatory cytokines and myeloperoxidase were also measured. The colonic motility was measured by upper GI transit in vivo and recorded using electrical stimulation organ bath technique in vitro. Freshly isolated smooth muscle from the rat colon were applied to determine the membrane potential and Ca2+‐ATPase activity, respectively. Key Results CBD or O‐1602 separately improved inflammatory conditions significantly in TNBS‐induced colitis rats. However, sole CBD pretreatment reduced GPR55 expression, which was up‐regulated in TNBS colitis. O‐1602 and CBD each lowered MPO and IL‐6 levels remarkably in TNBS colitis, while TNF‐α levels experienced no change. CBD rescued the downward colonic motility in TNBS colitis in vivo; however, it decreased the upward contraction of the smooth muscle strip under electrical stimulation in vitro. Pretreatment with CBD prevented against TNBS‐induced changes of Ca2+‐ATPase activity of smooth muscle cells. However, membrane potential of the smooth muscle cells decreased by TNBS experienced no change after O‐1602 or CBD import. Conclusions & Inferences The present study suggested that CBD participated in the regulation of colonic motility in rats, and the mechanisms may be involved in the regulation of inlammatory factors and Ca2+‐ATPase activity through GPR55.