Abstract Cannabinoid-1 (CB1) receptor activation affects gastrointestinal propulsion in vivo. It was our aim to further characterize the involved myenteric mechanisms in vivo and in vitro. In CB1−/− mice and wild-type littermates we performed in vivo transit experiments by charcoal feeding and in vitro electrophysiological recordings in mouse small intestinal smooth muscle. Ascending neuronal contraction (ANC) following electrical field stimulation was studied in rat ileum in a partitioned organ bath separating the aboral stimulation site from the oral recording site. The knockout animals displayed an accelerated upper gastrointestinal transit compared to control animals. The CB1 receptor antagonist AM251 stimulated the force of the ANC in a concentration dependent manner when added in the oral chamber. Anandamide significantly inhibited the ANC when added in the oral chamber. Neither AM251 nor anandamide had an influence on the contraction latency. No effects were observed when drugs were added in the aboral chamber, proving a CB1 mediated action on the neuromuscular junction. Resting membrane potentials and neuronal induced inhibitory junction potentials in CB1−/− mice were unchanged as compared to wild type. However, the electrophysiological slow waves were more sensitive to blockade of Ca2+ channels in CB1−/− mice. Our data strongly suggest a physiological involvement of the CB-1 receptor in the regulation of small intestinal motility. Therefore, CB1 receptors are a promising target for the treatment of motility disorders.