show Abstracthide AbstractHydroxyurea (HU) is an antitumor drug. However, HU exposure is associated with diminished ovarian reserve (DOR). Zi Chong granules, a Chinese Medicine, can protect against DOR, but little is known regarding its underlying mechanisms of DOR treatment, and thus the target of the present study. Female KM mice were randomly divided into three groups: the control group (Con), the hydroxyurea group (HU), and the Zi Chong group (ZC). The ovaries and uterus of mice were examined histologically by H&E. The levels of anti-Mullerian hormone (AMH), estradiol (E2), and progesterone (P) were quantified using ELISA kits. The number and quality of oocytes were assessed, and endometrial receptivity was evaluated by immunohistochemistry. 16S rDNA gene sequencing was used to analyze the composition and abundance of gut microbiome in feces, and non-targeted metabolomics was performed to detect serum metabolite profiles. Correlation analysis was performed to explore the relationships between different gut microbiota and differential metabolites. The results showed that ZC granules increased the number of primordial follicles in the ovaries, reduced excessive follicular atresia, restored low AMH, upregulated estrogen and progesterone secretion, and increased the number of mature oocytes after ovulation promotion. It also increased thickness of uterine endometrium and the number of glands, resulting in increased endometrial microvessel density (MVD), enhanced endometrial blood supply, reduced CD138 expression levels and endometrial inflammation. HU decreased the abundance of Lactobacillus spp. in mouse intestines and decreased arachidonic acid metabolism, tryptophan metabolism, spermidine and spermine biosynthesis, steroidogenesis and nicotinate and nicotinamide metabolism. Correlation analysis revealed that HU exerted its side effects by altering the gut microbiome and bacteria-derived metabolites, while ZC granules could reverse DOR partly depends on regulating gut microbiota and metabolites. Together, ZC granules may be a potential therapy for alleviating HU-induced DOR.