Objective To explore the characteristics and molecular mechanisms of ovarian function decline in naturally aging mice. Methods Eighteen female ICR mice of 2, 6, 10 and 14 months of age were included in the study, and the estrous cycle, ovarian index, pregnancy rate and embryo number were detected; the number of follicles was observed by HE staining; serum anti-Müllerian hormone (AMH) level was measured by ELISA; the protein and mRNA expression of P16, P21, proliferatoractivated receptor-γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) were detected by IHC and qPCR respectively, and the protein expression of PGC-1α and TFAM was detected by Western Blot; Spearman"s rank correlation analysis and logistic regression analysis were used to assess the associations among indicators. Results Compared with the 2M group, the percentage of estrous cycle disorders (P<0.05 in the 10M group, P<0.01 in the 14M group), ovarian index (P<0.05 in the 10M group, P<0.01 in the 14M group), serum AMH level (P<0.01 in the 10M group, P<0.01 in the 14M group), the number of embryos (P<0.01 in the 10M group, P<0.01 in the 14M group), and the pregnancy rate (P<0.01 in the 14M group) were significantly decreased; the number of follicles at all levels and the total number of follicles were decreased (P<0.01 in each group), and the number of atretic follicles (P<0.01 in each group) was increased; the expression of P16 and P21 mRNAs (P16: P<0.01 in the 10M and 14M groups; P21: P<0.05 in the 10M group, P<0.01 in the 14M group) and proteins (P16: P<0.01 in each group; P21: P<0.01 in the 10M and 14M groups) were elevated; Spearman"s correlation analysis showed that the age in months was negatively correlated with the ovarian index, serum AMH level, primordial follicle number, number of embryos, and the expression of PGC-1α and TFAM, and positively correlated with the expression of atretic follicle, P16, and P21 (all P<0.01); Logistic regression analysis showed that there was a significant negative correlation between the age in months and the pregnancy rate (P<0.01).PGC-1α mRNA was significantly decreased (P<0.01 in all groups), and TFAM mRNA was significantly decreased in the 10M and 14M groups (P<0.01); PGC-1α protein was significantly decreased in the 10M group (IHC: P<0.05; WB: P<0.01) and the 14M group (all P<0.01); TFAM protein was significantly decreased in the 6M, 10M, TFAM protein was significantly reduced in the 6M, 10M, and 14M groups (IHC: P<0.01; WB: P<0.01). Compared with the 2M group, the expression of PGC-1α and TFAM was positively correlated with ovarian index and serum AMH level, and negatively correlated with the expression of P16 and P21 (all P<0.01). Conclusions Ovarian function in mice declines progressively with months of age, as reflected by fewer follicles, reduced fertility, and up-regulation of aging markers, which may be associated with decreased expression of the mitochondrial biogenesis factors PGC-1α and TFAM.