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Here, the regenerative potential of menstrual blood-derived mesenchymal stem cells (MenSCs) was examined on restoration of premature ovarian failure (POF) ovaries in rats' POF model. Freshly isolated CD146 + MenSCs using magnetic-activated cell storing method were immediately injected into ovaries of POF rats. Four and eight weeks after cell administration, both ovarian tissues were sampled for histological examination and the expression of fibrosis-related genes. Serum samples were also prepared for hormonal analysis. At the endpoint, mating trials were performed to assess the fertility of POF rats following MenSC transplantation. Histopathological examination revealed the induction of POF after Ceftriaxone injection by increasing atretic follicles and abnormal morphologies. MenSCs transplantation increased the number of normal follicles and coincided with the reduction of follicular atresia. Biochemical analyses exhibited the reduction and increase of systemic follicle-stimulating hormone (FSH) and E2 respectively after MenSCs transplantation compared to the POF rats (P < .05). No significant differences in anti-mullerian hormone (AMH) blood levels were detected in this study between POF controls and MenSCs-treated rats. We noted moreover the transcriptional up-regulation of Smad 2, 4, and TGF-β1 in POF rats, and these values were decreased after MenSCs transplantation (P < .01). By contrast, the RNA expression of Smad 6 remained increased in both pre- and post-treatment with MenSCs groups (P < .05). Finally, we found an increase in neonate births in POF rats treated with MenSCs, and that this feature was associated with ovarian rejuvenation through amelioration of fibrosis. These data showed that MenSCs are promising cell lineage for the alleviation of POF in the rat model by controlling the fibrosis rate., (© 2021 John Wiley & Sons Ltd.)