The major involvement of chemokines and proteolytic enzymes has recently been discovered in the mobilization process. Here, we report that the degradation of BM stromal cell-derived factor (SDF-1) by matrix metalloproteinase (MMP)-9 is important in G-CSF-mediated hematopoietic stem/progenitor cells (HSPCs) mobilization. In this study, the SDF-1 concentration in healthy donors BM plasma decreased significantly after 5 days of G-CSF administration, with no obvious change of SDF-1 in the peripheral blood. We also observed a similar result by immunohistochemical staining on the BM biopsy slides. In vitro, mobilized BM plasma exhibited decreased chemotactic effect on CD34(+) cells, compared with steady-state BM plasma. MMP-9 protein and mRNA increased dramatically in the BM plasma in accordance with SDF-1 decrease. MMP-9 enriched supernatant from HT1080 cell-conditioned medium upregulated CXCR4 expression and the migration of BM CD34(+) cells toward SDF-1. SDF-1 was a substrate for MMP-9, furthermore, SDF-1 also stimulated MMP-9 proteolytic enzyme activity of BM CD34(+) cells, which facilitate HSPCs migration. In BALB/c mice, HSPCs mobilization was significantly inhibited by anti-SDF-1 antibodies or MMP inhibitor, o-phenanthroline. In conclusion, the degradation of BM SDF-1 by MMP-9 is a vital step in mobilization.