The effect of Na⁺-K⁺ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na⁺-K⁺ pump was activated by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM. In aortic rings, the Na⁺ ionophore monensin evoked EDR, and this EDR was inhibited by the NCX (reverse mode) inhibitor KB-R7943. Monensin-induced Na⁺ loading or extracellular Na⁺ depletion (Na⁺ replaced by Li⁺) increased [Ca²⁺]_i in MAECs and this increase was inhibited by KB-R7943. Na⁺-K⁺ pump activation inhibited EDR and [Ca²⁺]_i increase (K⁺-induced inhibition of EDR and [Ca²⁺]_i increase). The Na⁺-K⁺ pump inhibitor ouabain inhibited K⁺-induced inhibition of EDR. Monensin (>0.1 µM) and the NCX (forward and reverse mode) inhibitors 2,4-dichlorobenzamil (>10 µM) or Ni²⁺ (>100 µM) inhibited K⁺-induced inhibition of EDR and [Ca²⁺]_i increase. KB-R7943 did not inhibit K⁺-induced inhibition at up to 10 µM, but did at 30 µM. In current-clamped MAECs, an increase in [K⁺]_o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni²⁺ or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K⁺]_o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 µM), Ni²⁺ (300 µM), or KB-R7943 (30 µM). These results suggest that activation of the forward mode of NCX following Na⁺-K⁺ pump activation inhibits Ca²⁺ mobilization in endothelial cells, thereby modulating vasomotor tone.