The mechanisms responsible for prostate cancer metastasis are incompletely understood at both the cellular and molecular levels. In this regard, chemokines are a family of small, cytokine-like proteins that induce motility of neoplastic cells, leukocytes and cancer cells. The current study evaluates the molecular mechanisms of CXCL12 and CXCR4 in prostate cancer cell migration and invasion. We report that functional CXCR4 is significantly expressed by prostate cancer cell lines, LNCaP and PC3, when compared with normal prostatic epithelial cells (PrEC). As measured using motility and invasion chamber assays, prostate cancer cells migrated and invaded through extracellular matrix components in response to CXCL12, at rates that corresponded to CXCR4 expression. Anti-CXCR4 antibodies (Abs) significantly impaired the migration and invasive potential of PC3 and LNCaP cells. CXCL12 induction also enhanced collagenase-1 (metalloproteinase-1 (MMP-1)) expression by LNCaP and PC3 cells. Collagenase-3 (MMP-13) was expressed by prostate cancer cells, but it was not expressed by PrEC cells or modulated by CXCL12. CXCL12 increased MMP-2 expression by LNCaP and PC3; however, MMP-9 expression was elevated only in PC3 cells after CXCL12-CXCR4 ligation. PC3 cells also expressed high levels of stromelysin-1 (MMP-3) after CXCL12 stimulation. CXCL12 also significantly increased stromelysin-2 (MMP-10) expression by LNCaP cells. Stromelysin-3 (MMP-11) was expressed by LNCaP cells, but not by PC3 or PrEC cells and CXCL12 induced PC3 MMP-11 expression. Membrane type-1 MMP (MMP-14) was not expressed by PrEC or LNCaP cells, but CXCL12 significantly enhanced MMP-14 expression by PC3 cells. These studies reveal important cellular and molecular mechanisms of CXCR4/CXCL12-mediated prostate cancer cell migration and invasion.