<p>Sickle cell anemia (SCA) is characterised by hemolysis, endothelial dysfunction, and vaso-occlusion. P-selectin is associated with cellular adhesion, while nitric oxide (NO) deficiency is linked to vascular injury. Variants in guanosine triphosphate cyclohydrolase (GCH1), a key enzyme in NO synthesis, may influence these pathways. This study aimed to evaluate the association between GCH1 polymorphism, NO levels, and P-selectin levels in patients with SCA. This cross-sectional, case-control study included newly diagnosed and follow-up patients with SCA, excluding those with recent blood transfusion, specific therapies or major comorbidities. Plasma P-selectin and NO levels were estimated by enzyme-linked immunosorbent assay (ELISA) and Griess assay, respectively. Hemolytic and biochemical markers were assessed, and GCH1 polymorphism was genotyped in 70 patients by Sanger sequencing. The study included 50 patients with SCA, 50 individuals with sickle cell trait and 50 healthy controls. Patients with SCA exhibited significantly higher P-selectin (<i>P</i> &lt; 0.001) and lower NO (<i>P</i> &lt; 0.001) compared with controls. NO correlated negatively with P-selectin (<i>r</i>=-0.82), LDH (<i>r</i>=-0.25) and total bilirubin (<i>r</i>=-0.31), and positively with haptoglobin (<i>r</i> = 0.62). The P-selectin showed a negative correlation with haptoglobin (<i>r</i>=-0.505) and homocysteine (<i>r</i>=-0.192), and a positive correlation with total bilirubin (<i>r</i> = 0.298). A G &gt; A transition in GCH1 (allele A frequency 10.7%) was identified but no significant association between P-selectin and NO was observed. Hemolysis-driven oxidative stress is associated with NO depletion and P-selectin elevation, reflecting endothelial dysfunction in SCA. Although GCH1 polymorphism (rs8007267) was present, it had no major effect. P-selectin and NO may serve as potential biomarkers of vascular dysfunction in SCA.</p>