Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of cells that negatively regulate the immune response during tumor progression, inflammation, and infection. In this study, through gene-expression analysis, we have identified a new marker, CD49d, which is expressed exclusively on CD11b+ Gr-1 dull/int. MDSCs. We have characterized two subpopulations of MDSCs based on CD49d expression in two different settings, a mouse model of inflammatory bowel disease and tumor-bearing mice. The CD49d+ subset of MDSCs was mainly monocytic and strongly suppressed Ag-specific T cell proliferation in an NO-dependent mechanism similar to Gr-1 dull/int. MDSCs. Alternatively, CD49d− cells were granulocytic and poorly inhibited T cell proliferation compared with CD11b+ Gr-1 high cells. Both mouse models showed preferential expansion of the granulocytic CD49d− subset. We suggest that CD49d can be used as an alternative marker for Gr-1 to differentiate between the subpopulations of MDSCs together with CD11b, which will ultimately help in understanding the mechanisms of immune suppression by MDSCs.