BACKGROUND: Premature ovarian insufficiency (POI) is a reproductive disorder characterized by amenorrhea, reduced estrogen levels, and elevated gonadotropins. Increasing evidence highlights the involvement of microRNAs, particularly miR-155-5p, in inflammation and apoptosis. This study aimed to evaluate the expression level of miR-155-5p and key folliculogenesis genes. Anti-Müllerian hormone(AMH) and growth differentiation factor 9 (GDF9) in the ovarian tissue of a cyclophosphamide-induced POI mouse model compared with healthy controls, alongside histopathological assessment and follicle quantification. METHODS: Sixteen female NMRI mice were randomly assigned to two groups (n = 8 each). The experimental group received intraperitoneal cyclophosphamide (120 mg/kg) to induce POI, while the control group received normal saline. Ovarian tissues were harvested for molecular and histological analyses. Total RNA was extracted and subjected to cDNA synthesis, and miR-155-5p, AMH, and GDF9 expression were quantified by QRT-PCR. For morphological evaluation, serial ovarian sections were stained with hematoxylin-eosin (H&E), toluidine blue, and Periodic Acid-Schiff (PAS) special stains. RESULTS: miR-155-5p expression was significantly upregulated in the POI group compared with controls (P < 0.001). AMH expression was markedly downregulated (P < 0.001) and GDF9 expression was significantly upregulated (P < 0.05). Histopathology revealed reduced healthy follicles, increased atresia, stromal disruption, and oocyte degeneration in the POI group. Serum estradiol decreased, while FSH levels increased. CONCLUSION: Upregulation of miR-155-5p was associated with ovarian histopathological alterations and changes in folliculogenesis-related gene expression in the cyclophosphamide-induced POI model. These findings identify miR-155-5p as a potential candidate biomarker of ovarian injury in this experimental setting. However, the present findings do not establish a causal or mechanistic role for miR-155-5p. Further functional studies, including gain- and loss-of-function experiments and target validation, as well as clinical studies, are required to determine its biological and clinical significance.