Estrogen regulates a variety of physiological processes, including mammary gland growth, morphogenesis of the mammary gland, proliferation and differentiation, and elevating the expression of milk proteins. Many nuclear phosphorylated proteins such as pStat5 and mTOR regulate milk protein synthesis. But the detail of milk protein synthesis controlled at the transcript level and posttranslational level is not well-known. To contribute to the understanding of the molecular mechanism underlying estrogen action on the dairy cow mammary epithelial cells (DCMECs), nuclear phosphorylated proteins regulated by estrogen in DCMECs were identified. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization/time of flight mass spectrometry were used to identify the changes of nuclear phosphorylated proteins in DCMECs treated with estrogen. Seven proteins were identified differentially up-expressed in DCMECs after 24-h estrogen exposure: including glycyl-tRNA synthetase, previously reported in milk protein synthesis of DCMECs, belonging to the class-II aminoacyl-tRNA synthetase family; proteins involved in other cellular functions, such as translation initiation factors, GTP-binding nuclear proteins, heat-shock proteins, and proteins belonging to ubiquitin-proteasome system. This screening reveals that estrogen influences the levels of nuclear phosphorylated proteins of DCMECs which opens new avenue for the study of the molecular mechanism linking to milk synthesis.