<p>Cytoophidia are filamentous structures composed of CTP synthase (CTPS) and were first identified in the ovarian cells of <i>Drosophila</i>. As a highly conserved, membraneless organelle present across all three domains of life, cytoophidia exhibit dynamic behaviors essential for cellular homeostasis and function. Previous studies have demonstrated that cytoophidia are actively transported from nurse cells to the oocyte, suggesting a potential role in <i>Drosophila</i> oogenesis; however, the molecular and cellular mechanisms governing cytoophidium dynamics remain poorly understood. In this study, we employ live-cell imaging to systematically characterize the spatiotemporal dynamics of cytoophidia and to investigate the underlying regulatory mechanisms. Our findings reveal that cytoophidium dynamics depend on key cytoskeletal components, including microtubules, microfilaments, and myosin II. Disruption of either microtubules or microfilaments results in the disassembly or depolymerization of macro-cytoophidia, underscoring the essential role of the cytoskeleton in maintaining cytoophidium integrity and facilitating proper assembly. Collectively, these results establish that microtubules, microfilaments, and myosin II are pivotal for regulating cytoophidium dynamics. This study provides novel insights into the mechanisms of cytoophidium transport and assembly, and lays a foundation for further investigation of their functional significance in <i>Drosophila</i> oogenesis.</p>