Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency ofCTCs and lack of appropriate models. Here, we describe the characterization of a novelCTC-derived breast cancer cell line, designatedCTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+) breast cancer, resistant to endocrine therapy.CTC-ITB-01 remainedER(+)in culture, and copy number alteration (CNA) profiling showed high concordance betweenCTC-ITB-01 andCTCs originally present in the patient with cancer at the time point of blood draw.RNA-sequencing data indicate thatCTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductalPDXmouse model mirrored the clinical progression ofER(+)breast cancer. DownstreamERsignaling was constitutively active inCTC-ITB-01 independent of ligand availability, and theCDK4/6 inhibitor Palbociclib strongly inhibitedCTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to studyCTCbiology.