Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin) pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.