Objective To discuss the clinical efficacy of Ovary-Warming Pot (OWP) on premature ovarian insufficiency (POI) mice by regulating ovarian mitochondrial function and to provide a scientific basis for its clinical application. Methods Eight of 40 8-week-old female C57BL/6 were selected as blank group, and the rest were used to establish the POI models. Then 32 successfully modeled mice were randomly divided into model group, low-, medium- and high-dose OWP groups, with 8 mice in each group. The model group and blank group were given 0.01mL/g normal saline by gastric lavage. The general condition, body weight, and estrous cycle of mice were observed. After 3 weeks of administration, the wet ovarian weight of mice in each group was weighed to calculate the ovarian index. The serum anti-Müllerian hormone (AMH) level was determined by enzyme-linked immunosorbent assay (ELISA), the morphological changes of ovarian tissue were observed by hematoxylin-eosin (HE) staining, and the morphology and structure of mitochondria were observed by transmission electron microscopy. Western blot was used to determine the expression levels of optic atrophy 1 (OPA1) and PTEN-induced protein kinase 1 (PINK1). Results Compared with blank group, the mice in model group had less food intake and dull hair color; their growth rate of body mass was lower (P<0.05); the ovarian index was significantly lower (P<0.01); the estrous cycle was disordered; the serum AMH was lower (P<0.05); the growth follicles at all levels decreased, while atresia follicles increased significantly; the mitochondrial structure was seriously damaged, with obvious cavitation; the expression levels of OPA1 and PINK1 were significantly down-regulated (P<0.01). Compared with model group, the growth rates of body mass in high-, medium- and low-dose OWP groups were higher (P<0.05); the ovarian indexes in medium- and low- dose OWP groups were higher, and the estrus cycle became regular gradually; the serum AMH in medium- and high- dose OWP groups was significantly higher (P<0.05), the mitochondrial structure of mice oocytes was significantly improved, and the OPA1 and PINK1 protein expressions were significantly up-regulated (P<0.01). Conclusion Mitochondrial dysfunction may be one of the mechanisms leading to ovarian insufficiency in mice. However, OWP can regulate mitochondrial function and improve premature ovarian insufficiency, which may be related to the dynamic process of promoting mitochondrial self-renewal.