Because endothelial cells are fundamental to the maintenance of the functional integrity of the vascular wall, endothelial modifications in altered gravity conditions might offer some insights into the mechanisms leading to circulatory impairment in astronauts. We cultured human endothelial cells in a dedicated centrifuge (MidiCAR) to generate hypergravity and in two different devices, namely the Rotating Wall Vessel and the Random Positioning Machine, to generate hypogravity. Hypogravity stimulated endothelial growth, did not affect migration, and enhanced nitric oxide production. It also remodeled the actin cytoskeleton and reduced the total amounts of actin. Hypergravity did not affect endothelial growth, markedly stimulated migration, and enhanced nitric oxide synthesis. In addition, hypergravity altered the distribution of actin fibers without, however, affecting the total amounts of actin. A short exposure to hypergravity (8 min) abolished the hypogravity induced growth advantage. Our results indicate that cytoskeletal alterations and increased nitric oxide production represent common denominators in endothelial responses to both hypogravity and hypergravity.