Tumor cells disseminate to distant organs mainly through blood circulation, where they experience considerable levels of fluid shear flow. However, its influence on circulating tumor cells remains less understood. This study elucidates the effects of hemodynamic shear flow on biophysical properties and functions of breast circulating tumor cells with metastatic preference to brain. Only a small subpopulation of tumor cells are able to survive in shear flow with enhanced anti-apoptosis ability. Compared to untreated cells, surviving tumor cells spread more on soft substrates that mimic brain tissue but less on stiff substrates. They exhibit much lower expression of F-actin and cell stiffness but generate significantly higher cellular contractility. In addition, hemodynamic shear flow upregulates the stemness genes and considerably changes the expression of the genes related to brain metastasis. The enhanced cell spreading on soft substrates, reduced stiffness, elevated cellular contractility, and upregulation of the stemness and brain metastasis genes in tumor cells after shear flow treatment may be related to breast cancer metastasis in soft brain tissues. Our findings thus provide the first piece of evidence that hemodynamic shear flow regulates biophysical properties and functions of circulating tumor cells that are associated with brain metastasis, suggesting that tumor cells surviving in blood shear flow may better reflect the characteristics of organ preference in metastasis.