To investigate the association of eNOS gene polymorphism with essential hypertension in the Chinese Han population, we examined polymorphisms of the rs2070744 (T→C), rs1800780 (A→G), and rs3918181 (A→G) loci. The results demonstrated that the genotypic frequency at the rs1800780 (A→G) locus was significantly different between patients with essential hypertension and the control cohorts (P < 0.05); while genotypic frequencies and allelic frequencies at rs2070744 (T→C) and rs3918181 (A→G) loci had no statistical difference between the patient group and controls (P > 0.05). In addition, haplotype analysis found a statistically significant difference for haplotype TGA, with OR (95% CI) of 1.549 (1.116-2.150) (P < 0.05). These findings suggest that polymorphism of rs1800780 (A→G) in the eNOS gene may be one of the most important genetic factors associated with essential hypertension susceptibility, and those who have haplotype TGA may be at risk to develop essential hypertension.