Previous studies have demonstrated that an increase in dietary NaCl (salt) intake stimulated endothelial cells to produce transforming growth factor-β (TGF-β). The intent of the present study was to determine the functional significance of increased TGF-β on endothelial cell function. Young Sprague-Dawley rats were fed diets containing 0.3 or 8.0% NaCl for 2 days before treatment with a specific inhibitor of the TGF-β receptor I/activin receptor-like kinase 5 kinase, or vehicle for another 2 days. At day 4 of study, endothelial phosphorylated Smad2 (S465/467) increased and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) levels decreased in the high-salt-treated rats. In addition, phosphorylated Akt (S473) and phosphorylation of the endothelial isoform of NO synthase (NOS3) at S1177 increased. Treatment with the TGF-β receptor I/activin receptor-like kinase 5 inhibitor reduced Smad2 phosphorylation to levels observed in rats on the low-salt diet and prevented the downstream signaling events induced by the high-salt diet. In human umbilical vein endothelial cells, reduction in PTEN levels increased phosphorylated Akt and NOS3. Treatment of macrovascular endothelial cells with TGF-β1 increased phosphorylated NOS3 and the concentration of NO metabolites in the medium but had no effect on either of these variables in cells pretreated with small interfering RNA directed against PTEN. Thus, during high salt intake, an increase in TGF-β directly promoted a reduction in endothelial PTEN levels, which in turn regulated Akt activation and NOS3 phosphorylation. This effect closes a feedback loop that potentially mitigates the effect of TGF-β on the vasculature. © 2013 American Heart Association, Inc.