Evidence from ground-based animal studies using tail-suspended hindlimb unloaded rats model has clearly demonstrated that simulated microgravity-induced smooth muscle cell phenotype conversion, a characteristic vascular structural and functional remodeling, may be one of the key contributors to postspaceflight orthostatic intolerance. However, the rats model involves multiple collective effects of microgravity including cephalic fluid shift and postural muscle unloading on smooth muscle cells (SMCs). It cannot isolate a single factor from the collective ones and therefore is not ideal to study the effects of gravitational vector alteration alone on SMCs. To test the hypothesis that gravitational vector alteration per se might affect smooth muscle cell phenotype, a roller culture apparatus was employed to expose cultured rat aortic smooth muscle cells (RASMCs) to simulated microgravity. Cell proliferation, cell cycle distribution, apoptosis, migration, and nitric oxide production rates were measured and compared between the control and the simulated microgravity groups. Cell cytoskeleton reorganization induced by simulated microgravity was observed by confocal microscopy. Specific contractile and synthetic Gene expression at the mRNA level was quantified by reverse transcriptional polymerase chain reaction. It was observed that simulated microgravity suppressed RASMC proliferation and migration, enhanced cell apoptosis, stimulated NO release, and destroyed the original well-organized cytoskeleton. Moreover, at the mRNA level, long-time exposure (≥ 72 h) to simulated microgravity induced a contractile phenotype tendency by up-regulating smMHC expression. All these findings suggest that the phenotype modulation of vascular smooth muscle cells may be gravity dependent.