Acotiamide hydrochloride hydrate (acotiamide-HH) is the first approved drug in the world for the treatment of patients with functional dyspepsia in Japan. A statistically significant increase in the incidence of endometrial adenocarcinoma was found in a 104-week carcinogenicity study in rats, in a non-dose-dependent manner, and it was considered that further evaluation was required to clarify this issue. Therefore, we performed a uterotrophic bioassay using immature female rats, which is mentioned in the Organization for Economic Co-operation and Development (OECD) Guideline 440, to evaluate the effect of acotiamide-HH on estrogen, which is one of the most important mechanisms causing increase in the incidence of endometrial adenocarcinoma. The positive control substance selected was 17α-ethinyl estradiol (EE). While EE caused a dose-dependent increase in uterine weight, no increase in uterine weight, histopathological changes, or endometrial proliferation activity were observed in the acotiamide-HH treatment groups at doses of up to 1000 mg/kg. Based on this result, we concluded that acotiamide-HH has no potential risk to cause imbalance of the sex hormone environment in female rats.