The primary cilium plays an important role in mechanosensation in mammalian cells. To understand mechanosensation in the primary cilium, we combined a microfluidic device with super-resolution microscopy to study the primary cilium phenotypes. The microfluidic system enabled the precise control of the flow shear within a well-confined cell-culture environment. In addition, in situ cilia fixation was possible by switching from the culture medium to the fixation buffer instantaneously, which preserved the real-time cilium phenotype under the flow shear. After fixation, multiple cilium-specific proteins were immunostained to quantify the cilia bending behavior. We found that >50% of the primary cilia of mouse inner medullary collecting duct cells were highly aligned with the direction of flow under 11 Pa shear stress. Finally, we used super-resolution microscopy to observe the redistribution of two major cilium-specific proteins under flow shear, acetylated alpha-tubulin, and intraflagellar transport protein 88. To the best of our knowledge, this is the first platform to combine a microfluidic device with super-resolution microscopy to enable flow stimulation and in situ fixation for the observation of ciliary protein. This system can potentially be applied to the future development of a stimulation-enabled organ-on-a-chip to observe the intercellular signaling of primary cilia or for the analysis of disease mechanisms associated with ciliary mutations at the organ level.