Mesenchymal stem cells (MSCs), the multipotent progenitor cells, are sensitive to fluid shear stress (FSS). MSCs can migrate through the blood stream by intravasation into the circulatory system to transfer to distant positions through the blood stream. During the transferring process, MSCs may differentiate into cells of corresponding tissues for repair, or remain undifferentiated and initiate ectopic tissue formation, lipid accumulation, or calcification, which are closely related to the pathology of atherosclerosis. However, how the MSCs sense and respond to vascular FSS stimulation and lead to subsequent biological effects remains elusive. In this study, by using an in situ time-lapse microfluidic cell culture and observation system, we found that rat mesenchymal stem cells (rMSCs) presented a contraction and re-spread (CRS) process when they were initially subjected to a physiological FSS (1.3 Pa). Our subsequent studies demonstrated that integrin and cilia played key roles in sensing FSS. Calcium, F-actin, and Rho-kinase were key molecules in the mechanotransduction of the CRS of the rMSCs. Our study revealed the immediate response of the rMSCs to FSS. It will be helpful for the understanding of MSC-related tissue repair and the role of MSCs in the initiation of atherosclerosis.