This study aims to investigate the effects of the fat mass and obesity-associated gene (FTO) on the expression of lipid metabolism-related genes and triglyceride (TAG) synthesis in mammary epithelial cells (DMECs) of dairy goats through overexpression and interference techniques. The complete coding sequence (CDS) of the FTO gene in the mammary tissue of diary goats (Capra hircus) was cloned, and an FTO overexpression vector was constructed. Additionally, small interference RNA (siRNA) targeting FTO were designed and synthesized. RT-qPCR was used to detect changes in the expression of lipid metabolism-related genes and triglyceride content in DMECs after overexpressing or interfering with the FTO gene. The results indicated that the full length of the FTO gene CDS region in dairy goat was 1 518 bp, with sequence homology of 99%, 97% and 96% to sheep (Ovis aries) (NM_001104931.1), cattle (Bos taurus) (NM_001098142.1) and buffalo (Bubalus bubalis) (XM_006043050.4), respectively. Overexpression of FTO in DMECs significantly upregulated the mRNA levels of sterol-regulatory element binding protein 1 (SREBF1), fatty acid synthase (FASN), and diacylglycerol O-acyltransferase 2(DGAT2) (P<0.05), while significantly downregulating the expression levels of triglyceride hydrolysis-related genes such as hormone-sensitive lipase (HSL) (P<0.05). Furthermore, the TAG content in the cells significantly increased. Conversely, interference with FTO in DMECs yielded opposite results. In conclusion, FTO gene plays an important role in regulating lipid metabolism in DMECs of dairy goats, providing a theoretical basis for improving the regulatory network of lipid metabolism in mammary gland of dairy goat.