Abstract: Difenoconazole is widely used and has been found to have excessive residues in edible agricultural products and has been detected in serum and follicular fluid of infertile women. To investigate the effects of subacute oral exposure to difenoconazole on ovarian function, the changes of progesterone (P) and estradiol (E₂) in rat serum were determined after gavage administration of 50, 100, and 200 mg/(kg bw • d) of difenoconazole corn oil solution, and the number of follicles was counted by light microscopy after hematoxylin-eosin (HE) staining of rat ovaries. The results showed that the ovarian organ coefficients of rats exposed to each dose group and the control group were not significantly different. Serum progesterone levels in rats exposed to 200 mg/(kg bw • d) were significantly higher than those in the control group (P < 0.01), while those exposed to 50 and 100 mg/(kg bw • d) were not significantly different from the control group, but were significantly lower than those in the 200 mg/(kg bw • d) exposure group (P < 0.05). There was no significant difference in serum estradiol levels between the exposed and control groups. In terms of follicle development, the percentage of growing follicles was significantly higher in the 200 mg/(kg bw • d) exposed group than in the control group (P < 0.05), while there was no significant difference in the 50 and 100 mg/(kg bw • d) exposed groups compared to the control group. The research demonstrated that subacute oral exposure to difenoconazole at a dose of 200 mg/(kg bw • d) resulted in certain adverse effects on rat ovaries, leading to increased serum progesterone levels and growth follicle ratios.