〔Abstract〕 To explore the molecular mechanism of the therapeutic effect of total timosaponin on central precocious puberty (CPP) rats based on the RF-amide-related Peptide 3 (RFRP-3)/G protein-coupled receptor 147 (GPR147) signaling pathway. Methods Twenty-six day-old female SD rats were randomly assigned into a control group, a model group, an RFRP-3 up-regulated group (1.5 mg/kg), a total timosaponin group (200mg/kg), a total timosaponin+empty load group, and a total timosaponin+RFRP-3 knockdown group, with 12 rats in each group. A CPP rat model was prepared by subcutaneous injection of N-methyl-DL-aspartic acid (NMA) (40mg/kg). After modeling and administration, the opening time of the rat’s vulva and the first estrus interval daily were observed. Administration was stopped when the first estrus interval appeared in the model group. The day of vaginal opening and the time of the first estrus interval of rats were recorded in each group of rats. The wet masses of both ovaries and uterus were measured, and the ovarian and uterine indices were calculated; HE staining was used to observe the morphological changes of rat ovarian and uterine tissues, the number of corpus luteum in the ovaries and the thickness of the uterine wall were determined; ELISA method was applied to detect levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), and estradiol (E2); RT-qPCR was used to detect the mRNA expression of hypothalamic gonadotropin-releasing hormone (GnRH), KISS-1, GPR54, RFRP-3, and GPR147. Results Compared with the control group, the day of vaginal opening and the time of the first estrus interval of rats in the model group were advanced (P<0.05), the ovarian index and uterine index, number of ovarian corpus luteum and uterine wall thickness, serum LH, FSH, E2 levels, and the GnRH, KISS-1, and GPR54 mRNA expression levels in the hypothalamus increased (P<0.05), while the RFRP-3 and GPR147 mRNA expression levels decreased (P<0.05). Compared with the model group, the day of vaginal opening and the time of the first estrus interval of rats in the RFRP-3 up-regulated group and the total timosaponin group were delayed (P<0.05), the ovarian index and uterine index, number of ovarian corpus luteum and uterine wall thickness, serum LH, FSH, E2 levels, and the GnRH, KISS-1, and GPR54 mRNA expression levels in the hypothalamus decreased (P<0.05), while the RFRP-3 and GPR147 mRNA expression levels increased (P<0.05). Knocking down RFRP-3 could prominently weaken the delaying effect of total timosaponin on the development of precocious puberty in CPP rats (P<0.05). Conclusion Total timosaponin may inhibit the release of GnRH and premature activation of the hypothalamic pituitary gonadal axis by activating the RFRP-3/GPR147 signaling pathway, thereby alleviating the degree of precocious puberty development in CPP model rats.