Circulating tumor cells (CTCs) play a vital role in the metastasis and recurrence of breast cancer. CTCs are highly heterogeneous at the stage of Epithelial-to-Mesenchymal Transition (EMT), but the phenotypic and biological characteristics in different EMT stages remain poorly defined. We conducted an orthotopic mouse (4T1) model of breast cancer to isolate CTCs and identified two phenotypes of CTCs: intermediate E/M and mesenchymal CTCs. MTT, Colony formation, Transwell migration and invasion assays were utilized to examined cell proliferation, colony forming, migration and invasion ability. Both the intermediate E/M and mesenchymal CTCs exhibited lower rates of proliferation, colony formation and invasion, as compared to primary tumor cells. The mesenchymal CTCs had a higher rate of invasion but lower rates of proliferation and colony formation than the intermediate E/M CTCs. They also exhibited lower rates of growth and metastasis than the primary tumor cells in vivo, but the mesenchymal CTCs had a higher rate of metastasis than the intermediate E/M CTCs. Fluid shear stress induced the EMT transition of CTCs. The comprehensive analysis of CTCs proteomics discovered proteins that differentially expressed in the two types of CTCs and their primary tumor cells.