The aim of the present study was to evaluate the levels of pro-insulin and pro-insulin/ insulin ratio (PIR) in pregnant with normal glucose tolerance (NGT), pregnant with gestational diabetes mellitus (GDM) and women after delivery with GDM history. Normal pregnancy is characterized by progressive insulin resistance, which is physiologically compensated by an increase in insulin secretion. The higher secretion of the insulin precursor pro-insulin has been associated with β-cell dysfunction. A total of 102 pregnant women between 24-28 gestational weeks (53 GDM pregnant, 49 with NGT) and 22 postpartum with GDM history, as assessed by a 2h oral glucose tolerance test, were included in the study. Fasting plasma insulin and pro-insulin (PI) concentrations at the basal state were measured in all women. The ratio pro-insulin/insulin was calculated. BMI was significantly higher in GDM pregnant compared to NGT weight-matched group (30.56 ± 6.9 vs. 30.56 ± 6.9; p < 0, 011) and compared to the levels after delivery (30.56 ± 6.9vs. 27.9 ± 6, 27; p < 0, 001). Significant differences in the levels of PI between NGT and GDM pregnant (3.94 ± 2.78 vs. 7.59 ± 5.27; p = 0.006), between GDM and postpartum women (7.59 ± 5.27 vs. 4.46 ± 1.14; p = 0.022) were established. No signifi cant difference in the level of PIR between two pregnant groups was observed. Separately NGT and GDM showed signifi cant difference compared to young mothers (0.41 ± 0.14 vs. 0.148 ± 0.031, p < 0.02; 0.46 ± 0.16 vs. 0.148 ± 0.031, p = 0.009). Fasting insulin was statistically higher in GDM pregnant compare to NGT and women after delivery (13.84 ± 8.43 vs. 11.35 ± 7.38, p = 0.02; 13.84 ± 8.43 vs. 10.60 ± 7.53, p < 0.01). The correlation between PIR and BMI in the three groups studied were r = 0.416; r = 0,741; r = 0,556 (with statistical significance p = 0.01 between NGT and GDM pregnancy, p = 0.02 between GDM pregnancy and postpartum, p < 0.0001 between NGT pregnancy and young mother with GDM history). In our study, comparison of PI levels between pregnant with NGT and GDM demonstrated that the OR of developing GDM was 1.194 (95% CI, 1.028-1.329, P = 0.001). Increasing the value of PI with 10 pmol/l increases the risk for development of GDM with 19.4%. According to our results, pregnant with GDM have elevated levels of pro-insulin and PIR which could serve as a markers for this condition. These results support our findings about relationship and influence of BMI on β-cell functions, established in this study with normotolerant, gestational diabetes pregnant and women postpartum with GDM history. These results demonstrate that gestational diabetics have abnormalities in pancreatic beta-cell secretion, which are likely to be important both in the etiology of gestational diabetes and non-insulin dependent diabetes.