INTRODUCTION: Premature ovarian insufficiency (POI) is characterized by impaired ovarian function before 40 years of age and is associated with menstrual disturbance, infertility, hypoestrogenic symptoms, psychological distress, and long-term health risks. Hormone replacement therapy (HRT) is recommended for symptom control and prevention of estrogen-deficiency-related complications but does not reliably restore ovarian activity or fertility potential. Acupuncture and moxibustion are used as complementary interventions for ovarian dysfunction; however, evidence regarding their independent and combined adjunctive effects in women with POI remains limited. METHODS: This prospective, multicenter, assessor-blinded, 2 × 2 factorial randomized controlled trial will recruit 144 women with POI from three clinical centers. Participants will be randomly assigned in a 1:1:1:1 ratio to estradiol/dydrogesterone alone, acupuncture plus estradiol/dydrogesterone, moxibustion plus estradiol/dydrogesterone, or combined acupuncture and moxibustion plus estradiol/dydrogesterone. The intervention period will last 12 weeks. The primary outcome is the change from baseline to week 12 in antral follicle count (AFC). Secondary outcomes include serum anti-Müllerian hormone (AMH), sex hormones, ovarian hemodynamic parameters, menopausal and anxiety symptoms, Traditional Chinese Medicine syndrome scores, and menstrual status. Clinical pregnancy will be analyzed as an exploratory reproductive outcome during treatment and follow-up at weeks 24 and 48. Factorial models will estimate the main effects of acupuncture and moxibustion, whereas the acupuncture × moxibustion interaction will be considered exploratory. CONCLUSION: This trial will evaluate the adjunctive effects of acupuncture and moxibustion on AFC and related secondary clinical outcomes in a selected subgroup of women with POI receiving standard HRT. The factorial design will permit estimation of the independent effects of both interventions and exploratory assessment of their potential interaction. CLINICAL TRIAL NUMBER: https://itmctr.ccebtcm.org.cn/, identifier ITMCTR2025002408.