Chemokine CXCL12 is proposed to promote multiple steps in growth of primary tumors and progression to metastatic disease in more than 20 different cancers. Functions of CXCL12 previously were believed to be controlled only by receptor CXCR4, but CXCR7 was recently identified as a second receptor for this chemokine. CXCR7 increases tumor formation and metastasis in mouse models, suggesting that this receptor may also be a key target for blocking effects of CXCL12 in cancer. To image activation of CXCR7 in intact cells and living mice, we tested the hypothesis that binding of chemokine ligands to CXCR7 recruits beta-arrestins, a family of cytosolic adapter proteins that interact with many activated chemokine and related seven-transmembrane receptors. Using firefly luciferase protein fragment complementation, we established that chemokine ligands CXCL12 and CXCL11 significantly increase association of CXCR7 and beta-arrestins with preferential interaction of the receptor with beta-arrestin 2. The magnitude of interactions between CXCR7 and beta-arrestin 2 increased over time after treatment with ligands, contrasting with transient association of beta-arrestin 2 and CXCR4. beta-Arrestin 2 increased uptake of CXCL12 in cells expressing CXCR7, emphasizing the functional relevance of the interaction between CXCR7 and beta-arrestin 2. In an orthotopic xenograft model of human breast cancer, we used bioluminescence imaging to quantify changes in the association of CXCR7 and beta-arrestin 2. These studies demonstrate ligand-dependent interactions of CXCR7 with beta-arrestin 2 that promote accumulation of chemokines and establish an imaging assay for the dynamic regulation of CXCR7 by chemokines and candidate therapeutic agents in cell-based assays and living mice.