Phosphatidylcholine cytidine transferase α (CCTα) is a key rate-limiting enzyme in the CDP-choline pathway, the primary pathway for phosphatidylcholine (PC) synthesis in mammals. This study investigated the role of CCTα in lipid droplet (LD) formation, phospholipid synthesis, LD fusion, and lipophagy in bovine mammary epithelial cells (BMECs) through CCTα gene knockout (CCT-KO) and overexpression (CCT-OE). CCTα mRNA expression was significantly increased in bovine mammary gland tissue after lactation. In BMECs, CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on LD surfaces in the presence of linoleic acid. Compared with normal BMECs (NC), CCTα knockout (CCT-KO) cells had significantly greater LD diameters (1.53 μm vs. 1.68 μm,