Low-temperature environments in cold regions pose a significant threat to cattle farming. Bovine mammary epithelial cells (BMECs) are highly sensitive to cold stress, and acute cold stress can induce apoptosis, adversely affecting lactation performance and health. To explore the mechanism of acute cold stress-induced apoptosis in BMECs, we established an in vitro acute cold stress model. Results showed that mRNA levels of HSP90 increased significantly in a time-dependent manner after 2 h of cold stress, confirming successful model establishment. Following 4 h of cold stress, pro-apoptotic genes (Caspase-3, Bax) exhibited significantly elevated mRNA levels, while the anti-apoptotic gene (BCL-2) showed significantly reduced mRNA levels. Concurrently, the apoptosis rate increased significantly, indicating that acute cold stress induces apoptosis and suggesting the 4 h mark may represent a critical transition point. Integrated transcriptomic and functional analyses identified ENO1 as a core metabolic regulator counteracting acute cold stress-induced apoptosis in BMECs. As a multifunctional protein, ENO1 (alpha-enolase) acts as a central enzyme in glycolysis while exerting additional roles in cellular signaling and apoptotic processes, thereby participating in various pathophysiological regulations. Both mRNA and protein levels of