The reflux of gastric content from the nasopharynx into the middle ear via the Eustachian tube may disrupt inner ear functions. The purpose of this study was to investigate the effect of experimental gastric reflux on the cochlear function of rats. Twelve rats were included in this study. Acidified gastric pepsin was prepared by the addition of HCl and deionized water to pepsinogen from porcine stomach. The left ears were designated as the experimental ears, and the solution was delivered daily for 30 days. The control ears received an equal amount of a saline solution. Distortion product otoacoustic emissions were recorded at baseline (before the injection) and on days 3, 10, and 30. When the mean baseline distortion product otoacoustic emission measurements were compared with the final mean measurements on day 30, the acidified gastric pepsin caused statistically significant (P < 0.05) hearing loss in the experimental ears. There was no significant change in the hearing of the control ears. This is the first study on the ototoxicity of acidified gastric pepsin. Our results demonstrate that acidified gastric pepsin causes hearing loss due to inner ear ototoxicity in a rat animal model.