https://doi.org/10.1016/j.ccmp.2022.100068 Get rights and content Under a Creative Commons license open access Abstract Background The prevalence of premature ovarian insufficiency (POI) is gradually increasing, safer and more effective treatments are urgently needed. Objective The aim of this study was to evaluate the effects of Ningxin-Yishen formula (NXYSF) on D-galactose-induced POI mice as well as to shed a light on its potential mechanisms. Methods Six to eight weeks old female C57BL/6 mice were used in this study and randomly divided into six groups: control group; model group; estradiol valerate (EV) treatment group and NXYSF treatment group with graded doses (9.5, 19, and 38 g·kg?1/d). Both EV and NXYSF treatments were initiated at the 15th day of modeling and lasted for 28 days. Afterwards, the ovarian function was evaluated in each group by analyzing the proportion of primordial follicles as well as the serum sex hormone levels. Furthermore, network pharmacology approach was performed to elucidate the potential targets of NXYSF, which was verified through western blotting experiments finally. Results NXYSF could significantly reverse the inefficiency of weight gain caused by POI, and promote the development of primordial follicles. In addition, it could restore the abnormal serum anti-Müllerian hormone (AMH), estradiol (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Moreover, some crucial key gene targets including TP53 were as propose to be relate with the effect of NXYSF through network pharmacology analysis. Last but importantly, western blotting experiments confirmed that NXYSF could inhibit the expression of p53 protein in mouse ovaries. Conclusion Our findings proposed that NXYSF might be an effective alternative treatment for POI.