Metastasis is a highly inefficient process, with the vast majority of disseminated cancer cells failing to form secondary tumors. To establish brain metastases, cancer cells must traverse the blood-brain barrier and rapidly adapt to the specialized neural microenvironment. Here, we identify aminopeptidase N (CD13) as a pro-metastatic factor that supports the early stages of brain metastatic colonization. CD13 is upregulated in cancer cells during brain metastasis in both mouse models and human samples. CD13 knockdown significantly delayed metastatic growth and prolonged survival in mouse brain metastasis models while having no comparable effect on primary tumors. Loss- and gain-of-function experiments further revealed that CD13 enhances cancer cell seeding in the brain microenvironment. Together, these findings establish CD13 as an important mediator of brain metastatic colonization and a potential therapeutic target to prevent or delay disease progression.