CONTEXT: WDJ-S4, a standardized herbal formula, has been prescribed for refractory functional dyspepsia (FD) in Korea, but the detailed mechanisms are lacking. OBJECTIVE: The present study investigates the acceleration of gastrointestinal (GI) motility by WDJ-S4 and its potential mechanisms. MATERIALS AND METHODS: For five days, WDJ-S4 (50, 100 and 200 mg/kg) or mosapride (3 mg/kg) was orally given to BALB/c mice. After 20 h of fasting, loperamide (10 mg/kg, i.p.) was given to the mice except for normal group. To assess gastric emptying or intestinal propulsion, 500 μL of 0.05% phenol red or 200 μL of 5% charcoal diet was given once orally. RESULTS: Loperamide delayed gastric emptying and intestinal propulsion, while WDJ-S4 ameliorated peristaltic dysfunction, evidenced by reductions of remaining phenol red in the stomach and a marked increase of charcoal propulsion in the intestine. WDJ-S4 also normalized levels of acetylcholine and acetylcholine-related enzymes, including choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in the gastric antrum and jejunum. C-kit level and smooth muscle contraction-related genes were elevated by WDJ-S4 in both the gastric antrum and jejunum. CONCLUSION: Overall, WDJ-S4 can effectively promote GI motility. The efficacy is associated with modulation of acetylcholine pathway and the interstitial cells of Cajal (ICCs) activation. All the results provide scientific evidence supporting the clinical usage of WDJ-S4 for FD.