Objective To evaluate the effects of diabetes mellitus, diabetic retinopathy and degree of blood glucose (BG) regulation on retinal nerve fiber layer (RNFL) thickness by using a scanning laser polarimeter (NFAGDx). Methods We prospectively assessed RNFL thickness in four groups of patients, who were all age matched. Diabetic patients without diabetic retinopathy were grouped according to their BG regulation level into two, as: BG-regulated group (BG Ͻ140 mg/dl, HbA1c Ͻ8%, fructosamine Ͻ285 mol/l, TG Ͻ200 mg/dl, n = 50), and BG-non-regulated group (BG = 140-250 mg/dl, HbA1c Ͼ8%, fructosamine Ͼ285 mol/l, TG Ͼ200 mg/dl, n = 44). A group of patients with nonproliferative diabetic retinopathy (NPDR) formed the 3rd group (n = 41). The 4th group consisted of healthy subjects and acted as a control group (n = 50). Symmetry, superior maximum, ellipse modulation and the average thickness variables of NFA-GDx were used for the assessment. ANOVA test was used for the statistical analysis of variables between groups. Results The mean superior maximum and ellipse modulation values were statistically significantly lower than the control group in BG-non-regulated and NPDR groups (P Ͻ 0.05). The average thickness value was also statistically significantly lower than the control group in NPDR group. These values in the BG-regulated group were not statistically significantly different from the control group (P Ͼ 0.05). Conclusions This is the first clinical study demonstrating the effects of diabetic glucose regulation level on RNFL by using NFAGDx. RNFL thickness was seen to decrease with development of diabetic retinopathy and with impairment of metabolic regulation. This issue should be taken into account while assessing RNFL in diabetic glaucomatous patients.