As an extracellular matrix protein, osteopontin (OPN) has been shown to play an important role in regulation of the immune response to tumors. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid progenitors, are major components of the immune suppressive tumor microenvironment and contribute to tumor evasion of the immune response. However, the specific regulating mechanisms underlying MDSCs expansion remain unclear. Here, we found that MDSCs accumulated in the spleen and tumors of 3LL tumor-bearing mice. Supernatant collected from 3LL cells was able to induce the expansion of MDSCs in peripheral blood mononuclear cell (PBMC) in vitro. Results of enzyme linked immunosorbent assay showed high levels of OPN and matrix metalloproteinase-9 (MMP-9) in this supernatant. Silencing OPN can effectively reduce MDSCs frequency in vivo and in vitro. Furthermore, a specific fragment of OPN, OPN-32 kDa cleaved by MMP-9 was detected in the supernatant from 3LL cells. Overexpression of OPN-32 kDa in 3LL cells induced MDSCs expansion. Inhibition of MMP-9 by monoclonal antibody and inhibitor (TIMP-1) reduced MDSCs expansion in vitro and in vivo. These findings suggest that the MMP-9-cleaved OPN fragment, OPN-32kDa, was responsible for MDSCs expansion, which may contribute to tumor's evasion of the immune response.