Ephedrae Herba (EH) has been used in Asian traditional herbal medicine to cure bronchial asthma, cold, flu, chills, fever, headache, nasal congestion, and cough. In this study, we evaluated the subchronic toxicity of an Ephedrae Herba aqueous extract (EHAE) in male and female F344 rats. The EHAE was administered orally daily at doses of 0, 125, 250, 500, and 1000 mg/kg bw/day for 13 weeks. Toxicological assessment was performed to determine mortality, clinical signs, and changes in body weight, food consumption, ophthalmological, urinary, hematological, and serum biochemical parameters, macroscopic and microscopic evaluations, and organ weights. We found that oral administration of EHAE to F344 rats for 13 weeks resulted in histopathological changes in the kidneys and salivary glands. In the kidneys, increased incidence and severity of tubular basophilia were observed in females administered 1000 mg/kg bw/day of the extract. In the salivary glands, acinar cell hypertrophy was observed in males administered 500 mg/kg bw/day and in both sexes administered 1000 mg/kg bw/day of the extract. All test article-treated groups of males and females administered ≥250 mg/kg bw/day showed increased absolute and relative salivary gland weights. Therefore, the NOAEL (No Observed Adverse Effect Level) was determined as 125 mg/kg bw/day for both sexes of rats under the present experimental conditions.