Aim: Gestational diabetes mellitus (GDM) is a glucose intolerant condition that affects 14% of all pregnancies. Diabetes mellitus (DM) occurs in 30 - 70% of patients with GDM after delivery. DM and GDM are associated with structural and functional deterioration of the renovascular system. Our aim is to investigate the association Glu-298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene with serum nitric oxide levels and microalbuminuria in patients with GDM and healthy pregnancies. Material and methods: Serum nitric oxide (NO) levels, urinary excretion of albumin and Glu298Asp polymorphism of the eNOS gene were analyzed in 68 patients with GDM and 73 healthy controls. High performance liquid chromatography (HPLC-Griess) method was used to analyze serum NO levels. Microalbuminuria was evaluated by rate nephelometry method. The Glu298Asp polymorphism of the eNOS gene was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: Nitric oxide, glucose, creatinine, and microalbuminuria were significantly different between the patients and the control subjects (p = 0.001, p = 0.001, p = 0.002, and p = 0.005, respectively). There was a significant difference between groups in terms of the ratio of GG/GT+TT of eNOS gene Glu298Asp (p = 0.02). The patients with GT+TT genotype had significantly higher microalbuminuria levels and lower NO concentrations (22.16 vs. 9.51, p = 0.005, and 10.56 vs. 12.73, p = 0.021, respectively). The presence of T allele of eNOS gene is an independent predictor of microalbuminuria (OR: 2.346, 95% confidence interval: 1.247 - 5.238, p = 0.02) as well as serum glucose and NO concentration. Conclusion: The G894T polymorphism of eNOS gene and decreased NO concentration seem to be independent predictors of increased urinary excretion of albumin in patients with GDM. Determining the frequency of eNOS gene G894T polymorphism may help to identify pregnancies at increased risk of microalbuminuria.