Obesity induced by high-fat diet (HFD) is a multi-factorial disease including genetic, physiological, behavioral, and environmental components. Drosophila has emerged as an effective metabolic disease model. Cytidine 5'-triphosphate synthase (CTPS) is an important enzyme for the de novo synthesis of CTP, governing the cellular level of CTP and the rate of phospholipid synthesis. CTPS is known to form filamentous structures called cytoophidia, which are found in bacteria, archaea, and eukaryotes. Our study demonstrates that CTPS is crucial in regulating body weight and starvation resistance in Drosophila by functioning in the fat body. HFD-induced obesity leads to increased transcription of CTPS and elongates cytoophidia in larval adipocytes. Depleting CTPS in the fat body prevented HFD-induced obesity, including body weight gain, adipocyte expansion, and lipid accumulation, by inhibiting the PI3K-Akt-SREBP axis. Furthermore, a dominant-negative form of CTPS also prevented adipocyte expansion and downregulated lipogenic genes. These findings not only establish a functional link between CTPS and lipid homeostasis but also highlight the potential role of CTPS manipulation in the treatment of HFD-induced obesity.