Background <p>Amoxicillin, a commonly used broad-spectrum penicillin antibiotic in pregnancy, has sparked controversy regarding its impact on fetal growth and development. There remains a lack of systematic research on the specific influence of prenatal amoxicillin exposure (PAmE) on the ovarian development of the offspring, as well as the precise " toxicity windows “.</p> Methods <p>We established PAmE mouse models at different stages [(gestational day, GD) 10–12, GD13-15 or GD16-18], doses (75, 150 or 300&#xa0;mg/kg·d), and courses (single/multiple courses). On GD18, fetal serum and ovaries were collected to assess changes in serum estradiol levels and evaluate ovarian morphology, pregranulosa cell function, and oocyte-related parameters.</p> Results <p>PAmE led to pathological damage in fetal mouse ovaries, characterized by disrupted germ cell cysts and reduced the number of germ cells. Cell proliferation was enhanced while apoptosis was reduced. Moreover, PAmE upregulated the expression of pregranulosa cell steroid synthesis-related genes (e.g., <i>Sf1</i>,<i> Star</i>,<i> P450scc</i>) in the fetal ovaries, particularly in the high-dose groups at all gestational stages. The expression of the oocyte marker gene Figlα increased in all PAmE groups, while follicle development-related genes (<i>Nobox</i> and <i>Bmp15</i>) were downregulated, particularly during early to mid-pregnancy and in the single-course exposure groups. Further investigation revealed that PAmE enhanced IGF1 expression in fetal ovaries and inhibited the Pten-Akt-Foxo3a signaling pathway.</p> Conclusions <p>Amoxicillin exhibits ovarian developmental toxicity, influencing fetal ovarian cell proliferation, apoptosis, pregranulosa cell estrogen synthesis, oocyte numbers, and follicle assembly. This study provides evidence guiding the rational use of amoxicillin in pregnancy and assessing potential ovarian development risks.</p> Graphical Abstract <p></p>