Azithromycin is widely used in treating bacterial infections during pregnancy. Previous studies suggest prenatal exposure (PAzE) induces embryonic developmental toxicity. However, the influence of PAzE on fetal adrenal gland development is unknown. Pregnant mice received azithromycin in varying ways: different stages (mid- and late-pregnancy), doses (50, 100, and 200 mg/kg d), and courses (single- and multi-course). Adrenal gland morphology, cell proliferation, apoptosis, steroid synthesis, and expression of key transcriptional factors were examined. PAzE predominantly affected fetal adrenal gland development in males, characterized by obvious pathological changes (irregular arrangement and decreased density of adrenocortical cells, aggravated cytoplasmic vacuolization), weakened cell proliferation (decreased Pcna but increased Caspase3 expression), and inhibited steroidogenesis (reduced expression of Star, 3β-hsd, P450c21, and P450c11). The most significant damage occurred with multi-course high-dose (clinical dose) azithromycin treatment in late-pregnancy, possibly linked to inhibited Cited2 expression. This study delineated the sex-specific toxic effects of PAzE on fetal adrenal gland development, influenced by various stages, doses, and courses of azithromycin treatment. These findings contribute to a better understanding of azithromycin's safe use during pregnancy and offer a crucial theoretical and experimental foundation for future research.