This study aimed to screen the optimal active fraction of Artemisia rupestris L. water extract and its different polar extracts for promoting gastrointestinal motility, while investigating its pharmacological effects and underlying mechanisms. Gastric emptying rate and intestinal propulsion rate in mice were measured using chromatic marker method, combined with in vitro smooth muscle experiments to compare and identify the most active fraction among water extract, ethyl acetate extract, n-butanol extract, and residual aqueous fraction. An L-arginine-induced gastrointestinal motility disorder mouse model was established. Serum motilin (MTL) and gastrin (GAS) levels were detected by ELISA. Liquid chromatography-mass spectrometry (LC-MS) was employed for chemical component identification of active fractions, with subsequent correlation analysis between pharmacodynamic results and active constituents. Results demonstrated that the n-butanol extract contained higher total flavonoids (131.4 ± 5.59 mg/g) compared to other fractions. This extract significantly reduced gastric pigment retention rate (P < 0.01) and enhanced intestinal propulsion rate (P < 0.01) in normal mice. In the motility disorder model, it effectively alleviated symptoms and elevated serum MTL and GAS levels (P < 0.05). In vitro experiments confirmed its synergistic effect on acetylcholine-induced contraction and antagonistic action against atropine-induced relaxation. Fourteen chemical components were identified, predominantly flavonoids including characteristic constituents such as quercetin and kaempferol. In conclusion, the n-butanol extract of A. rupestris likely regulates gastrointestinal motility through flavonoid-mediated modulation of cholinergic pathways and gastrointestinal hormone secretion, representing a potential mechanism for its prokinetic effects.