Morphological, functional, and gene expression studies have established the phenotypic plasticity of smooth muscle cells (SMCs). These cells have been shown to respond to environmental stimulants such as extracellular matrix and growth factors. Cellular changes can vary between extremes defined as the contractile and synthetic states. Various growth factors have been shown to have profound effects on the phenotype of these cells. In this study, we intended to investigate the effects of growth factor-rich medium on the protein expression of vascular SMCs in culture. Interestingly, transiently changing the type of medium did not result in any apparent morphological differences, yet we hypothesized that some cellular factors might still be altered. In order to understand what kind of intracellular molecular changes should be expected to occur during the medium change, we analyzed global protein expression changes using nano-LC-MS/MS in smooth muscle cell cultures that were isolated and grown in one medium formulation and temporarily switched to the other. Our data indicate that proteins playing a role in energy metabolism (glycolysis), translation, and folding of proteins are affected, along with regulatory molecules and cytoskeletal proteins. The individual proteins and their significance are discussed within the scope of this paper. ; Morphological, functional, and gene expression studies have established the phenotypic plasticity of smooth muscle cells (SMCs). These cells have been shown to respond to environmental stimulants such as extracellular matrix and growth factors. Cellular changes can vary between extremes defined as the contractile and synthetic states. Various growth factors have been shown to have profound effects on the phenotype of these cells. In this study, we intended to investigate the effects of growth factor-rich medium on the protein expression of vascular SMCs in culture. Interestingly, transiently changing the type of medium did not result in any apparent morphological differences, yet we hypothesized that some cellular factors might still be altered. In order to understand what kind of intracellular molecular changes should be expected to occur during the medium change, we analyzed global protein expression changes using nano-LC-MS/MS in smooth muscle cell cultures that were isolated and grown in one medium formulation and temporarily switched to the other. Our data indicate that proteins playing a role in energy metabolism (glycolysis), translation, and folding of proteins are affected, along with regulatory molecules and cytoskeletal proteins. The individual proteins and their significance are discussed within the scope of this paper.