Gastroesophageal reflux disease (GERD) is a common disease, in which the reflux of gastric acid causes mucosal damage of the esophagus and/or troublesome symptoms. Esomeprazole, a proton pump inhibitor, has been used for treatment of GERD in Japan since 2011; namely, only little is known about its effect on gastric acid secretion in Japanese. We, therefore, assessed the relationship between dose and timing of esomeprazole administration and gastric acid inhibition in 11 healthy male Japanese volunteers by directly examining gastric acid secretion capacity. In this randomized, open-label, three-way crossover study, the subjects were dosed with esomeprazole 10 mg or 20 mg once a day (q.d.), or 20 mg twice a day (b.i.d.) for 14 days, and pentagastrin-stimulated gastric acid secretion was measured by endoscopic gastrin test. At steady states, gastric acid inhibition rates were significantly higher in esomeprazole 20 mg b.i.d. (median 100.0%, interquartile range [IQR] 99.4-100%, P = 0.027) or 20 mg q.d. (100.0%, IQR 99.7-100%, P = 0.016), compared with 10 mg q.d. (98.4%, IQR 84.4-100%). At trough states, esomeprazole 20 mg b.i.d. showed significantly higher gastric acid inhibition (99.6%, IQR 99.0-100%) than did 20 mg q.d. (84.2%, IQR 76.4-88.8%, P = 0.002) or 10 mg q.d. (64.9%, IQR 59.1-76.7%, P = 0.001). Thus, esomeprazole 20 mg b.i.d. was sufficient to inhibit > 99% gastric acid secretion in healthy subjects. We propose that esomeprazole 20 mg b.i.d. is effective for treating Japanese patients with refractory GERD who require long-lasting gastric acid inhibition.