PURPOSE: Metastasis is responsible for most cancer-related deaths; hence, therapies designed to minimize metastasis are greatly needed. The precise cellular and molecular mechanisms used by cancer cells for metastasis are not fully understood; however, the metastatic spread of neoplastic cells is probably related to the ability of these cells to migrate, invade, home, and survive locally. The migration of tumor cells shares many similarities with leukocyte trafficking, which is regulated by chemokine receptor-ligand interactions. The current study evaluates the molecular mechanisms of CCL25 and CCR9 in prostate cancer cell migration and invasion. EXPERIMENTAL DESIGN: In the current study, real-time quantitative polymerase chain reaction, flow cytometry analysis, and in vitro migration as well as invasion chamber analysis (with and without antibody-mediated inhibition) were used to ascertain the biological and functional significance of CCR9 expression by normal prostatic epithelial cells (PrEC) or prostate cancer cell lines (LNCaP-10995 and PC3). RESULTS: We report that functional CCR9 is highly expressed by LNCaP cells and modestly, yet significantly, expressed by PC3 cells when compared with PrEC cells. Neutralization of CCL25-CCR9 interactions impaired the migration and invasion potential of the LNCaP and PC3 cell lines. CCL25 differentially modulated the expression of collagenase-1 or matrix metalloproteinase (MMP)-1, collagenase-3 (MMP-13), stromalysin-2 (MMP-10), stromalysin-3 (MMP-11), and gelatinase-A (MMP-2), but not MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, or MMP-14 in prostate cancer cells. CONCLUSIONS: These studies suggest that the expression and activation of CCR9 affect cancer cell migration, invasion, and MMP expression, which together may affect prostate cancer metastasis.