Tumor-associated macrophages (TAM) play a critical role in promoting tumor development and metastasis. In the present study, we found that legumain, an asparaginyl endopeptidase, was highly expressed on the surface of TAM. A doxorubicin-based prodrug specifically activated by legumain selectively ablated TAM and resulted in a significant reduction of angiogenic factors and related tumor vessel growth. Treatment with the prodrug also suppressed circulating tumor cells and myeloid immune suppressor Gr-1+/CD11b+ cells in tumor-bearing animals. After selective ablation of TAM using the prodrug, tumor growth and metastases were greatly inhibited in murine tumor models. These results indicate that legumain-activated prodrugs targeting TAM in tumors might represent a novel anticancer strategy.