Introduction The ability of mesenchymal stromal cells (MSC) to suppress T‐cell function has prompted their therapeutic use for graft‐versus‐host disease (GVHD) control. However, as MSC also modulate the activity of NK cells, which play an important role in graft‐versus‐leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity. Objective The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM‐CSM) or adipose tissue (AT‐MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL. Methods Human NK cells were stimulated with IL‐15 in the presence of BM‐MSC or AT‐MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays. Results Although both BM‐MSC and AT‐MSC induced a similar inhibition of NK cell proliferation, only BM‐MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN‐γ secretion. Conclusion These results suggest that, in the context of GVHD inhibition, the use of AT‐MSC rather than BM‐MSC could further preserve NK cell activity and, thus, favor GVL.