Background In vitro study showed that NADPH oxidase ( NO x), the most important enzyme producing reactive oxygen species ( ROS ), plays a role in the process of platelet activation. However, it is unclear if changes in its activity affect platelet activation in vivo. Methods and Results In vivo and ex vivo experiments assessing platelet activation were investigated in healthy subjects, obese patients, and subjects with different low rates of NO x2 activity, namely X‐linked chronic granulomatous disease (X‐ CGD ) patients and X‐ CGD carriers. We included 27 X‐ CGD patients, 31 women carriers of hereditary deficiency of NO x2, 31 obese women, and 62 healthy subjects matched for sex and age. Plasma levels of soluble sCD40 L ( sCD 40L) and soluble P ( sP) ‐selectin, 2 markers of in vivo platelet activation, were reduced in X‐CGD patients ( sCD 40L=−55%; sP ‐selectin=−51%, P <0.001) and in X‐CGD carriers ( sCD 40L=−41%; sP ‐selectin=−57%, P <0.001) compared with respective controls. Conversely, obese women, who disclosed NOx2 upregulation, had significantly higher plasma levels of sCD 40L (+47%, P <0.001) and sP ‐selectin (+70%, P <0.001) compared with controls. Ex vivo study showed platelet isoprostane downexpression and enhanced platelet NO generation in both X‐CGD patients and X‐CGD carriers compared with controls; opposite findings were observed in obese patients. Correlation analysis showed that platelet NOx2 regulation was directly associated with plasma levels of sCD 40L ( R =0.336, P <0.001) and sP ‐selectin ( R =0.441; P <0.001). Conclusions The study provides the first evidence that in vivo platelet activation is significantly and directly associated with NO x2 activity. Platelet NO x2 may be a novel target for platelet activation inhibition.