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ETHNOPHARMACOLOGICAL RELEVANCE Bu Shen Huo Xue Tang (BSHXT) is a traditional Chinese medicine formula that is clinically used in the treatment of premature ovarian insufficiency (POI). However, its therapeutic mechanism remains unclear. AIM OF THE STUDY This study aimed to investigate the underlying molecular mechanism of pharmacological activity of BSHXT, via the Nrf2/Keap1 signaling pathway, in the treatment of autoimmune POI. MATERIALS AND METHODS The chemical composition of BSHXT was analyzed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The autoimmune POI mouse model was induced by immunizing mice twice, with zona pellucida (ZP) glycoprotein 3 antigen. The autoimmune POI mice were continuously administered BSHXT for 28 days. Body weight and organ indices were recorded. The pathological morphology of the ovaries was observed. The estrous cycle of each mouse was recorded. Immunofluorescence assay was used to detect the levels of ZP antibodies in the mouse ovaries. The levels of ZP antibodies, follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), estradiol (E2), luteinizing hormone (LH), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured. The expression of genes and proteins involved in the Nrf2/Keap1 signaling pathway were measured by Q-PCR and IHC, respectively. RESULTS Twenty-one compounds were identified in the BSHXT water extract. BSHXT was found to increase the body weight and ovarian index, improve ovarian function, and reduce disorders in the estrus cycle. It also reduced the expression of ZP antibodies in the ovaries and serum of POI mice. BSHXT significantly increased E2 and AMH levels and decreased FSH and LH levels. It also increased the levels of SOD, and reduced MDA levels. The levels of Nrf2 and Keap1 were also increased, and the expression of Nrf2, HO-1 and NQO1 genes was upregulated. CONCLUSIONS BSHXT has a therapeutic effect on autoimmune POI in mice, which may be a result of the enhanced antioxidant capacity and activation of the Nrf2/Keap1 signaling pathway. BSHXT is a good drug candidate for use as a protective agent for POI and may be used in clinical practice.