Significance Early developmental exposure to endocrine active compounds causes late-stage effects and alterations in the reproductive tract of adult mice. Unexpectedly, estrogen receptor alpha (ERα) plays a pivotal role in mediating these developmental effects. As a model outcome from these developmental effects, we present transcriptome and DNA methylation profiling of the seminal vesicles (SVs) following neonatal diethylstilbestrol (DES) exposure. ERα mediates transcriptome aberrations in SVs of adult mice that impact developmental reprogramming at adulthood. DNA methylation dynamically changes during development, and methylation is greater in ERα knockout mice compared with wild type. Expression levels of DES-altered genes are associated with their DNA methylation status. These findings provide unique evidence for understanding the developmental actions and mechanisms of endocrine-disrupting chemicals in human health.