Objective: To explore the protective effect of mild moxibustion on the ovary of mice with premature ovarian insufficiency (POI) and its mechanism. Methods: SPF female ICR mice with normal estrus cycle were divided into blank group, model group, moxibustion group and moxibustion + Compound C group according to the random number table method, with 20 mice in each group. The mice in the blank group were given normal saline daily. The mice in the other groups were gavaged with Tripterygium wilfordii polyglycosides[75 mg/(kg·d)] for 14 consecutive days for modeling. An hour later, the mice in the moxibustion group were treated with mild moxibustion every day, and bilateral "shenshu" and "guanyuan" "zhongwan" were alternately applied every other day, and each point was treated with mild moxibustion for 10 minutes each day. Thirty minutes after gavaging Tripterygium wilfordii polyglycosides, the mice in the moxibustion + Compound C group were given intraperitoneal injection of Compound C (10 mg/kg); an hour later, mild moxibustion was applied same as the moxibustion group. The mild moxibustion was applied once a day for 14 consecutive days. The protective effect of moxibustion on ovary was evaluated by ovarian index, rate of estrus cycle disorder, ovarian tissue morphology, the number of follicles at all levels and serum sex hormone levels. Quantitative real-time polymerase chain reaction and immunohistochemistry were used to detect the mRNA and protein expression of adenosine 5′-monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) in ovarian tissue. Serum sex hormones oxidative stress markers, adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels in ovarian granulosa cells were detected by enzyme-linked immunosorbent assay, luciferase method and chemifluorescence method, respectively. Results: Compared with the model group, the ovarian index of the moxibustion group increased (P<0.05), the rate of estrous cycle disorder decreased (P<0.05), the number of atretic follicles decreased (P<0.05), and the number of primordial follicles, primary follicles, secondary follicles and antral follicles all increased (P<0.05). The levels of serum anti-Müllerian hormone and estrogen increased (P<0.05), while the level of follicle-stimulating hormone decreased (P<0.05); the expressions of protein and mRNA of AMPK and PGC-1α were upregulated (P<0.05); the activity of serum superoxide dismutase increased, the content of malondialdehyde decreased, and the ATP level of granulosa cells increased, while the mean fluorescence intensity of ROS decreased (P<0.05). However, when Compound C was given before moxibustion intervention, the protective effect on the ovary was significantly reduced, as shown by the reduction of ovarian index (P<0.05), the rate of estrus cycle disorder increased (P <0.05), the number of atretic follicles increased (P<0.05), and the number of primordial follicles, primary follicles, secondary follicles and antral follicles decreased (P<0.05). Conclusion Moxibustion can protect ovarian function in mice by promoting the activation of AMPK and PGC-1α signaling, inhibiting oxidative stress response and regulating hormone levels.