The cytoskeleton is an important structural component that regulates various aspects of cell morphology, movement, and intracellular signaling. It plays a pivotal role in the cellular response to biomechanical stimuli, particularly in endothelial cells, which are critical for vascular homeostasis and the pathogenesis of cardiovascular diseases. Mechanical forces, such as shear and tension, activate intracellular signaling cascades that regulate transcription, translation, and cellular behaviors. Despite extensive research into cytoskeletal functions, the precise mechanisms by which the cytoskeleton transduces mechanical signals remain incompletely understood. This review focuses on the role of cytoskeletal components in membrane, cytoplasm, and nucleus in mechanotransduction, with an emphasis on their structure, mechanical and biological behaviors, dynamic interactions, and response to mechanical forces. The collaboration between membrane cytoskeleton, cytoplasmic cytoskeleton, and nucleoskeleton is indispensable for endothelial cells to respond to mechanical stimuli. Understanding their mechanoresponsive mechanisms is essential for advancing therapeutic strategies for cardiovascular diseases.