Mammary glands in cows are highly dynamic, making genomic stability particularly crucial. Continuous lactation and self-renewal of these glands are primary contributors to genomic instability. Results: We employed transcriptomic and proteomic methods to analyze the expressional characteristics in the mammary glands of cows with varying levels of milk production. Our findings indicated differences in relevant pathways, including DNA damage repair and apoptosis, which are influenced by increasing parity. Notably, ATR protein levels in the mammary glands of low-yield dairy cows were reduced. Following in vitro silencing of ATR, β-galactosidase content increased in aging mammary epithelial cells, with cell cycle arrest in the G2 and S phases. Secretory phenotypes associated with aging, including IL-6, IL-10, IL-1β, INF-γ, and IL-2, were elevated, along with increased TNF-α content. The expressions of DNA repair-related proteins, including PIG3, PARP1, and Cleaved caspase3, were upregulated, and SP1 expression was decreased. Furthermore, the expressions of cytochrome C and BAK increased, and ATR silencing inhibited mTOR and STAT5 lactation signaling pathways, resulting in elevated STAT3 protein levels associated with mammary gland degeneration. Conclusions: This study emphasizes the significance of the ATR protein in the mammary glands of dairy cows, contributing valuable insights into maintaining their health and presenting a novel perspective on strategies to enhance their lifespan.