Erythropoietin (EPO) acts on erythroblasts in the bone marrow (BM) to stimulate the formation of red blood cells. In this study, we wanted to determine whether BM-derived mesenchymal stromal cells (MSCs) can be used as cellular vehicles to deliver EPO in mice without the use of viral vectors. After isolation and characterization of murine MSCs (mMSCs), different transient transfection procedures were compared for their efficacy of gene transfer of the pEGFP-N2 plasmid. Nucleofection outperformed magnetofection and lipofection. Stably transfected mMSCs were generated by selection with G418-disulfate and single-cell-colony-forming unit (sc-CFU) assays without changes in their proliferation capacity and osteogenic/adipogenic differentiation potential. Next, murine EPO was stably introduced into mMSCs by nucleofection of a plasmid encoding the epo and egfp genes. Intraperitoneal transplantation of EPO-expressing mMSCs increased serum EPO levels, hematocrit and hemoglobin of C57BL/6 mice within 1 week. The hematocrit remained elevated for 5 weeks, but production of antibodies against both transgenes was detected in the hosts and serum EPO levels normalized. Our results suggest that nonviral gene delivery into MSCs can be used to enhance the known beneficial effects MSCs by additional production of therapeutic factors like EPO in vivo.