Hemoglobin (Hb) may impact the transduction of endothelium‐dependent and nitric oxide (NO) mediated vasodilator activity, given its contribution to shear stress stimuli and diverse biochemical reactions with NO. We hypothesized that an acute reduction in [Hb] and hematocrit (Hct) would increase brachial artery flow‐mediated dilation (FMD). In eleven healthy males (28 ± 7 years; 23 ± 2 kg m−2), FMD (Duplex ultrasound), arterial blood gases, Hct and [Hb], blood viscosity, and NO metabolites (ozone‐based chemiluminescence) were measured before and after isovolumic hemodilution, where ∼20% of whole blood was removed and replaced with 5% human serum albumin. Hemodilution reduced Hct by 18 ± 2% (P < 0.001) and whole blood viscosity by 22 ± 5% (P < 0.001). Plasma nitrite (P = 0.01), S‐nitrosothiols (P = 0.03), and total red blood cell NO (P = 0.001) were collectively reduced by ∼15–40%. Brachial artery FMD increased by ∼160% from 3.8 ± 2.1 to 9.7 ± 4.5% (P = 0.004). Statistical covariation for the shear stress stimulus did not alter FMD, indicating that the increase in FMD was not directly related to alterations in whole blood viscosity and the shear stimulus. Collectively, these findings indicate that hemoglobin scavenging of NO appears to be an important factor in the regulation of FMD under normal conditions through constraint of endothelium‐dependent NO‐mediated vasodilation in healthy humans