Magnolol, as the main active ingredient of Traditional Chinese Medicine, can significantly improve gastrointestinal motility disorders (GMD). In the present study, metabolomics was used to investigate the mechanism of magnolol improving L-arginine induced GMD in rats. SD rats were randomly divided into control group, model group and magnolol treated group. L-arginine was injected intraperitoneally in model and magnolol groups to induce GMD model. All intervention regimens were administered by oral gavage, once a day for five consecutive days. Relative gastric emptying rate and propulsive intestinal rate were measured. Metabolites in serum were analyzed based on UPLC-MS metabolomics technique. Magnolol significantly promoted gastric emptying and small intestinal propulsion. Compared with the model group, the level of serotonin and L-tryptophan significantly reversed ( < 0.05) and 22 metabolites reversed in the magnolol group. According to MetPA database analysis, magnolol has mainly affected 10 major metabolic pathways which were related to each other, Tryptophan metabolism is the most critical metabolic pathway associated with gastrointestinal tract. These findings suggest that magnolol has a significantly promoting effect on L-arginine induced gastrointestinal motility disorder in rats, the mechanism is to reduce the production of nitric oxide to weaken the function of nitric oxide relaxing the gastrointestinal smooth muscle and increase the content of serotonin to promote gastrointestinal peristalsis and motility, secretion, absorption of nutrients.