Background: Autism spectrum disorder (ASD) is characterized by impairments in social communication and restricted or repetitive behaviors or interests. ASD is now diagnosed in more than one out of 100 children and is biased towards males by a ratio of at least 4:1. Many possible explanations and potential causative factors have been reported, such as genetics, sex, and environmental factors, although the detailed mechanisms of ASD remain unclear. Methods: The dams were exposed through oral contraceptives to either vehicle control (VEH) alone, levonorgestrel (LNG) alone, ethinyl estradiol (EE) alone, or a combination of LNG/EE for 21 days during their pregnancy. The subsequent 10-week-old offspring were used for autism-like behavior testing, and the limbic tissues were isolated for analysis. In another experimental group, 8-week-old male offspring were treated by infusion of ER beta overexpression/knockdown lentivirus in the amygdala, and the offspring were analyzed after 2 weeks. Results: We show that prenatal exposure of either LNG alone or a LNG/EE combination, but not EE alone, results in suppression of ER beta (estrogen receptor beta) and its target genes in the amygdala with autism-like behavior in male offspring, while there is a much smaller effect on female offspring. However, we find that there is no effect on the hippocampus and hypothalamus. Further investigation shows that ER beta suppression is due to LNG-mediated altered methylation on the ER beta promoter and results in tissue damage with oxidative stress and the dysfunction of mitochondria and fatty acid metabolism, which subsequently triggers autism-like behavior. Overexpression of ER beta in the amygdala completely restores LNG-induced ER beta suppression and autism-like behaviors in offspring, while ER beta knockdown mimics this effect, indicating that ER beta expression in the amygdala plays an important role in autism-like behavior development. Conclusions: We conclude that prenatal levonorgestrel exposure induces autism-like behavior in offspring through ER beta suppression in the amygdala. To our knowledge, this is the first time the potential effect of oral contraceptives on the contribution of autism-like behavior in offspring has been discovered.