Amino acids (AA) are one of the key nutrients that regulate cell proliferation and casein synthesis in cow mammary epithelial cells (CMEC), but the mechanism of this regulation is not yet clear. In previous reports, sestrin2 (SESN2) protein was found to respond to AA depletion resulting in negative effects on the mTORC1 pathway in human cells. In this study, the effect of SESN2 on AA-mediated cell proliferation and casein synthesis in CMEC was assessed. After 12 h of AA starvation, CMECs were cultured in the absence of all AA (AA-), in the presences of only essential AA (EAA+), or of all AA (AA+). Cell proliferation was texted by MTT method, expression of 4 kinds of caseins, SESN2, and the mammalian target of rapamycin complex 1 (mTORC1) pathway associated proteins in protein level was tested by Western blotting, and the lysosomal localization of mTOR was tested by immunofluorescence. The results showed that cell proliferation, casein expression, and activation of mTORC1 pathway were increased; but SESN2 expression was decreased in response to increased EAA or AA supply. Cells were transfected with SESN2 overexpressed or inhibited plasmid. Cell proliferation, expression of 4 kinds of caseins, SESN2 and mTORC1 pathway associated proteins in protein level, and the lysosomal localization of mTOR was tested. The results showed that cell proliferation, casein expression, and activation of the mTORC1 pathway were all controlled by SESN2 expression. Furthermore, the increase in cell proliferation, casein expression, and activation of the mTORC1 pathway in response to AA supply was inhibited by overexpressing SESN2, and those effects were reversed by inhibiting SESN2. These results indicate that SESN2 is an important inhibitor of mTORC1 in CMEC blocking AA-mediated cell proliferation and casein synthesis.