Recent studies have identified arterial stiffening as a predictor of some vascular diseases such as pulmonary hypertension, which is characterized by dysfunction of small arteries. Stiffening is shown to cause changes in blood flow, extending high pulsatile flow into small arteries that normally experience steady flow conditions. However, few studies have investigated the mechanisms underlying the effects of arterial stiffening on vascular remodeling. This paper develops a pulsatile flow system for the co-culture of vascular smooth muscle cells and endothelial cells and then characterizes the response of these cells to changing upstream stiffness and increased pulsatility. A flow chamber to house the cell co-culture was designed with the aid of computational flow dynamics and experimental tests. The purpose of the design process was to quantify the flow area to produce a uniform area where the cells are exposed to the same flow profile to ensure accurate results for flow wave specific analysis. This study demonstrated that the designed flow system produces uniform area for cellular assays providing a tool to enhance the understanding of vascular remodeling in the arterial stiffening circulations or pulsatility variation environments. With this system, contractile and proliferating protein expression of smooth muscle cell co-cultured with endothelial cell was examined. Smooth muscle cells were embedded into a collagen matrix with endothelial cells seeded on a porous membrane in direct contact with the collagen; flow was applied directly to the endothelial cells. Immunoflurescent staining and western blot protein assays quantified the contractile and proliferative proteins in the smooth muscle cells, while polymerase chain reaction examined contractile and proliferative genes released by the endothelial cells. Results show that vascular remodeling induced by high pulsatile flow caused increased contractile markers in the smooth muscle cells. The results demonstrates that increased flow pulsatility may increase the hypertrophy of smooth muscle cells in the medial layer of arteries, and arterial stiffening significantly affects downstream small arterial remodeling through hemodynamic mechanism as evident in pulmonary hypertension.