Abstract Clusters of circulating tumor cells (CTC) exhibit more robust metastatic properties than single CTC. Thus, understanding the distinct behaviors of CTC clusters and how CTC clustering is regulated may offer new insights into how to limit metastasis. In this study, we utilized an in vivo confocal system to observe the clustering behavior of CTC in real time, finding that the number of clusters increased proportionally with the growth of the primary tumor. Our experiments also indicated that the flow rate of the CTC clusters in blood vessels was relatively slower than single CTC due to increased vessel wall adhesion. Depending on disease stage, 5% to 10% of total CTC in circulation were in clusters, with this proportion increasing to >24% within lung metastases examined. Notably, in the 4T1 mouse model of breast cancer metastasis, we found that injecting host animals with urokinase-type plasminogen activator, a clinical thrombolytic agent, was effective at preventing the assembly of CTC clusters and prolonging overall host survival by approximately 20% relative to control animals. Our results suggest a tractable approach to limit metastasis by suppressing the formation or stability of CTC clusters circulating in the blood of cancer patients. Cancer Res; 75(21); 4474–82. ©2015 AACR.