Ethylene glycol monomethyl ether (EGME), which is widely used in various industrial products, is known for adverse effects on the reproductive system in adult rats. However, the effects of EGME on reproductive development in juvenile rats have not been demonstrated. In order to investigate the effects of EGME on the female reproductive system and pubertal development in juvenile rats, EGME was administered to female Sprague Dawley rats from postnatal day 21 to 41 at a dose level of 0, 50, 100, or 300 mg/kg. The animals were examined for general condition, body weight, vaginal opening (VO), estrous cyclicity, and histopathology of reproductive organs. EGME treatment resulted in a prolonged estrous cycle interval characterized by persistent diestrus at 50 mg/kg without effects on body weight, timing of VO, or histology of the reproductive organs. EGME at 100 mg/kg induced decreases in body weight gain, a delay of VO, and irregular estrous cycle with absence of corpora lutea and hypertrophy of uterine epithelium indicating disturbance of the ovulatory process associated with hormonal effect. At 300 mg/kg, there was significant delay of puberty due to severe growth retardation. The present results revealed that irregular estrous cycle is a first indicator of the effects of EGME on the female reproductive system in juvenile rats, with delayed pubertal onset and ovulatory process disturbance at a higher dose.