Objectives: Micro- and macrovascular endothelial deterioration has been mentioned in diabetic pregnants with critical clinical outcome for the fetus. Our aim was to measure serum and cord blood concentrations of methylated arginines in patients with gestational diabetes and find a relationship with endothelial dysfunction. Materials and methods: Methylated arginines were detected with high performance liquid chromatography mass spectrometry via electrospray ionization positive technique with a chromatographic C18 column. Results: Although gestational diabetes mellitus (GDM) groups' asymmetric dimethylarginine (ADMA) levels were higher compared to control group, this difference was not significant. Control cord blood ADMA and N-monomethylarginine levels were significantly higher than insulin-regulated GDM cord blood ADMA and N-monomethylarginine levels (p = 0.001; p = 0.003, respectively). Diet-regulated GDM group's cord blood N-monomethylarginine was significantly higher than insulin-regulated GDM group's cord blood N-monomethylarginine (p = 0.045). A negative correlation was found between cord blood symmetric dimethylarginine and oral glucose tolerance testing 0h glucose values (r = −0.453, p = 0.002). Conclusions: According to this study's results, methylated arginine levels may not be associated with endothelial deterioration in GDM otherwise with preeclampsia risk.