Endothelial cells (EC) respond to shear stress to maintain vascular homeostasis, and a disrupted response is associated with cardiovascular diseases. To understand how different shear stress modalities affect EC morphology and behavior, we developed a microfluidic device that concurrently generates three different levels of uniform wall shear stress (WSS) and six different WSS gradients (WSSG). In this device, human umbilical vein endothelial cells (HUVECs) exhibited a rapid and robust response to WSS, with the relative positioning of the Golgi and nucleus transitioning from a non-polarized to polarized state in a WSS magnitude- and gradient-dependent manner. By contrast, polarized HUVECs oriented their Golgi and nucleus polarity to the flow vector in a WSS magnitude-dependent manner, with positive WSSG inhibiting and negative WSSG promoting upstream orientation. Having validated this device, this chip can now be used to dissect the mechanisms underlying EC responses to different WSS modalities, including shear stress gradients, and to investigate the influence of flow on a diverse range of cells during development, homeostasis and disease.