Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein involved in a variety of cellular processes and plays a critical role in the regulation of gene expression. Recently， Tudor-SN was found to be upregulated in mammary epithelial cells during lactation in response to prolactin， which further to regulate milk protein synthesis. However， the detailed regulatory mechanism of Tudor-SN to milk protein still remains to be elucidated. In our study， we observed that the levels of Tudor-SN and phosphor-Tudor-SN (Thr103) were both enhanced upon prolactin stimulation. Immunofluorescence assays demonstrated that prolactin treatment facilitated the nuclear transport of Tudor-SN. Further study revealed that the phosphorylation of Tudor-SN was depended on activated JNK. Coimmunoprecipitation assays disclosed that Tudor-SN might be phosphorylated directly by JNK. Using gene mutation assays， we further discovered that mutation of Thr to Ala at site of 103 prevented the nuclear transport of Tudor-SN. Thus， these results suggested the essential mechanism of the activated Tudor-SN in milk protein regulation in response to prolactin， which may provide some new sights into improve milk protein production.