Tumor-associated factors are related to increased accumulation of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs). However, the exact mechanism of how genetic factors control the expansion of MDSCs in tumor-bearing hosts remains elusive. Herein, we found that tumor-associated MDSCs and their subsets, mononuclear MDSCs and polymorphonuclear MDSCs, have decreased expression of miR-223 when compared to CD11b+Gr1+ cells from the spleen of disease-free mice. With the differentiation of CD11b+Gr1+MDSCs from bone marrow cells (BMCs) upon exposure to tumor-associated factors, the expression of both pri-miR-223 and mature miR-223 was downregulated, indicating that the expression of miR-223 could be regulated by tumor-associated factors. Interestingly, miR-223 remarkably inhibits differentiation of BMCs into CD11b+Gr1+MDSCs in the presence of tumor-associated factors by targeting myocyte enhancer factor 2C (MEF2C). Using reconstituted s.c. tumor models, miR-223 also suppresses accumulation of CD11b+Gr1+MDSCs, whereas its targeting molecule MEF2C increases the number of MDSCs. Tumor growth is slower in mice infused by miR223-engineered BMCs than in mice infused with control transfected BMCs. As miR-223 and its target molecule MEF2C are highly conserved between mice and humans, the modulation of miR-223 in tumor-induced CD11b+Gr1+MDSCs may exert an important role in controlling the increased accumulation of CD11b+Gr1+MDSCs in patients with tumor.