Background Diethylstilbestrol (DES) has been shown to disrupt the morphology and proliferation of gubernaculum testis cells. Objective This study aimed to elucidate the mechanism underlying DES induced damage to gubernaculum testis based on proteome analysis. Results Neonatal mice were exposed to DES or control vehicle and the gonads were collected for isobaric tags for relative and absolute quantitation based proteomics analysis. We found that at the early and late stages of gubernaculum development, treatment with different concentrations of DES upregulated the expression of proteins which were generally involved in several function categories including single-organism process, cellular process, binding, catalytic activity, cell part and organelle. KEGG pathway analysis showed that cardiac muscle contraction, oxidative phosphorylation, calcium signaling and cGMP-PKG signaling pathway were the most dysregulated pathways associated with DES in the late stage of gubernaculum development, while steroid biosynthesis was the only enriched pathway in the early stage of gubernaculum development. Conclusion Proteomics profi ling and functional analysis reveal new clues on how DES interferes with cellular processes and disrupts the development of gubernaculum. These findings have potential applications in the treatment of genitourinary diseases such as cryptorchidism.