Background and purpose:Endocannabinoids (via cannabinoid CB1 receptor activation) are physiological regulators of intestinal motility and food intake. However, their role in the regulation of gastric emptying is largely unexplored. The purpose of the present study was to investigate the involvement of the endocannabinoid system in the regulation of gastric emptying in mice fed either a standard diet (STD) or a high‐fat diet (HFD) for 14 weeks.Experimental approach:Gastric emptying was evaluated by measuring the amount of phenol red recovered in the stomach after oral challenge; CB1 expression was analysed by quantitative reverse transcription‐PCR; endocannabinoid (anandamide and 2‐arachidonoyl glycerol) levels were measured by liquid chromatography‐mass spectrometry.Key results:Gastric emptying was reduced by anandamide, an effect counteracted by the CB1 receptor antagonist rimonabant, but not by the CB2 receptor antagonist SR144528 or by the transient receptor potential vanilloid type 1 (TRPV1) antagonist 5′‐iodoresiniferatoxin. The fatty acid amide hydrolase (FAAH) inhibitor N‐arachidonoyl‐5‐hydroxytryptamine (but not the anandamide uptake inhibitor OMDM‐2) reduced gastric emptying in a way partly reduced by rimonabant. Compared to STD mice, HFD mice exhibited significantly higher body weight and fasting glucose levels, delayed gastric emptying and lower anandamide and CB1 mRNA levels. N‐arachidonoylserotonin (but not rimonabant) affected gastric emptying more efficaciously in HFD than STD mice.Conclusions and implications:Gastric emptying is physiologically regulated by the endocannabinoid system, which is downregulated following a HFD leading to overweight.British Journal of Pharmacology (2008) 153, 1272–1280; doi:10.1038/sj.bjp.0707682; published online 28 January 2008