The effects of acotiamide on gastrointestinal motility have not been sufficiently investigated. The aim of this study was to determine whether single preprandial acotiamide or mosapride intake might affect the gastric emptying rate using the 13C breath test. Here, 11 healthy volunteers participated in a randomized three-way crossover study. The subjects received acotiamide (100 mg) or mosapride (5 mg) or placebo before liquid test meal ingestion. Gastric emptying was estimated by determining following parameters: the time required for 50% emptying of the labeled meal (T1/2), lag time for 10% emptying of the labeled meal (Tlag), gastric emptying coefficient (GEC) and regression-estimated constants (β and κ). These parameters were calculated from a 13CO2 breath excretion curve using conventional formulas. The acotiamide, mosapride and placebo conditions were compared, revealing that for gastric emptying rates (values expressed as median), T1/2 (87.83571 min vs 79.95057 min vs 88.74378 min, p = 0.1496), Tlag (46.36449 min vs 42.2897 min vs 47.08094 min, p = 0.4966), GEC (4.382027 vs 4.211441 vs 4.248495, p = 0.8858), β (1.917728 vs 1.757062 vs 1.869141, p = 0.4066) and κ (0.834051 vs 0.819820 vs 0.789523, p = 0.1225) did not significantly differ. In this study, acotiamide (100 mg) or mosapride (5 mg) had no effect on gastric emptying.