Acephate is an organophosphate foliar and soil insecticide that is used worldwide. In this study, the transgenerational ovarian developmental toxicity caused by acephate, along with its in utero reprogramming mechanisms, were explored. Thirty female virgin Wistar albino rats were randomly assigned to three groups: one control group and two acephate treatment groups. The treatment groups received daily low or high doses of acephate (34.2 mg/kg or 68.5 mg/kg body weight, respectively) from gestational day 6 until spontaneous labor, resulting in F1 offspring. At 28 days, a subgroup of F1 females were euthanized. The ovaries were extracted, thoroughly cleaned, and weighed before being fixed for further analysis. The remaining F1 females were mated with normal males to produce the F2 generation. The F1 female offspring presented reduced fertility and body weight, whereas the ovarian weight index and sex ratio increased in a dose-dependent manner. Structural analysis revealed altered follicular abnormalities with ovarian cells displaying pyknotic nuclei. Additionally, the gene and protein expression of Cyp19 decreased, whereas that of Gdf-9 increased in the high-dose treatment group (68.5 mg/kg). We also observed significantly increased expression levels of ovarian estrogen receptor 1 (Esr1) and insulin-like growth factor 1 (Igf1), whereas Insl3 expression was significantly decreased. The F2 female offspring presented reproductive phenotype alterations similar to those of F1 females including decreased fertility, reduced Cyp19 gene and protein expression, and structural ovarian abnormalities similar to those of polycystic ovary syndrome (PCOS). In conclusion, acephate induced ovarian developmental toxicity across two generations of rats, which may be linked to changes in the ovarian Cyp19, Gdf9, Insl3, and Igf1 levels.