Purpose To evaluate retinal nerve fiber layer (RNFL) thickness in patients with diabetes mellitus compared with age-matched normal control subjects, to assess the correlation between the RNFL thickness and the severity of retinopathy, and to investigate whether diabetes mellitus is a potential source of abnormal results in glaucoma screening or evaluation with scanning laser polarimetry (SLP). Design Cross-sectional analysis of normal and diabetic eyes. Methods setting: Institutional clinical study. patients: One hundred twenty-eight subjects with type 2 diabetes mellitus and 50 age-matched normal control subjects without glaucoma or glaucoma-suspect. All patients underwent imaging with SLP and repeatable automated perimetry. Subjects with diabetes mellitus were classified into four stages on the basis of the severity of retinopathy that was assessed by dilated funduscopic examination, high-quality fundus color photography, and fluorescein angiography. main outcome measures: The SLP (GDx VCC software, version 5.5.0) parameters. Results The RNFL thickness in patients with diabetes mellitus was reduced significantly compared with age-matched normal control eyes ( P < .01). Moreover, all RNFL thickness parameters of the GDx VCC software decreased significantly as an exacerbation of diabetic retinopathy ( P = .0019, P = .0045, P = .0010 for temporal-superior-nasal-inferior-temporal (TSNIT) average, superior average, inferior average, respectively). The nerve fiber indicator also increased significantly ( P < .0001), despite an absence of glaucomatous optic nerve damages. Conclusion The RNFL thickness in type 2 diabetes mellitus, which was measured by GDx VCC software, significantly decreased with the severity of diabetic retinopathy. The presence of diabetes mellitus can be a source of false-positive results and overestimation of glaucomatous optic neuropathy when eyes are screened with GDx VCC software.