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Background: This study aimed at determining the effect of the herbal mixture estrogen inhibition formula (EIF) and its possible mechanisms by precocious puberty animal models and network pharmacology-based analysis. Methods: Precocious puberty animal models were established by a single injection of 300 μg danazol, then female rats were administered EIF, vaginal openings were monitored, uterus and pituitary indices were determined. The levels of ALP, E2, LH, and FSH were measured using ELISA kits. Real-time PCR was performed to evaluate the mRNA expression of GnRH, UNC5C, and netrin-1 in hypothalamic tissues. We applied network pharmacological analysis to predict potential targets and pathways of EIF. Results: EIF delayed danazol-induced early vaginal opening. In the onset model, EIF reduced the increased levels of serum ALP, E2, LH, and FSH; as well as mRNA expressions of GnRH, Netrin-1, and UNC5C. Moreover, long-term administration of EIF not only diminished all impaired factors but also had no effect on the normal development of the animals. The gene set enrichment analysis showed that the targets of EIF are mainly associated with the GnRH signaling and ovarian steroidogenesis pathways. Conclusion: EIF could be used in preclinical research for the treatment of precocious puberty by the inhibition of HPGA pre-maturation.