Cells mobilized from the bone marrow can contribute to endothelial regeneration and repair. Nevertheless, cardiovascular diseases are associated with diminished numbers and function of these cells, attenuating their healing potential. Gene transfer of endothelial nitric oxide synthase (eNOS) can restore the activity of circulating cells. Furthermore, estrogen accelerates the reendothelialization capacity of early outgrowth cells (EOCs). We hypothesized that overexpressing eNOS alone or in combination with estrogen stimulation in EOCs would potentiate the beneficial effects of these cells in regulating smooth muscle cell (SMC) function. Native human EOCs did not have any effect on human coronary artery SMC (hCASMC) proliferation or migration. Transfecting EOCs with a human eNOS plasmid and/or stimulating with 17β-estradiol (E2) increased NO production 3-fold and enhanced EOC survival. Moreover, in co-culture studies, eNOS overexpressing or E2-stimulated EOCs reduced hCASMC migration (by 23% and 56% respectively), vs. control EOCs. These effects do not implicate ERK1/2 or focal adhesion kinases. Nevertheless, NOS-EOCs had no effect on hCASMC proliferation. These results suggest that overexpressing or activating eNOS in EOCs increases their survival and enhances their capacity to regulate SMC migration through paracrine effects. These data elucidate how eNOS overexpression or activation in EOCs can prevent vascular remodeling.