Objectives: To estimate serum nitric oxide (NO) and asymmetric dimethylarginine (ADMA) levels in pregnant women and correlate it with severity of gestational diabetes mellitus (GDM) judged by Oral Glucose Tolerance Test (OGTT). Patients & Methods: 225 pregnant women gave fasting blood samples to estimate baseline blood glucose (BG), and serum insulin, NO and ADMA, and to undergo OGTT. Insulin resistance (IR) was indicated by HOMA-IR score >2. At 24th week, blood sample were re-evaluated using OGTT and women were categorized in Study (GDM) and Control (free of GDM) group. Results: At 24th week, HOMA-IR scores were significantly higher than baseline scores and in study versus control women. Baseline serum ADMA levels were significantly higher, while serum NO levels were significantly lower in study versus control women and correlated with body mass index (BMI), GDM, BG and HOMA-IR score of all women. Serum ADMA and NO showed significant correlation with extent of serum insulin change, but low serum NO is significant predictor for development and severity of IR. Conclusion: IR induces endothelial dysfunction manifested as low serum NO and high serum ADMA. Baseline serum NO could predict the extent of serum insulin change.