Gestational diabetes mellitus (GDM) is a common complication with negative impacts on mother and child. The primary aim of this study was to examine whether plasma glucose cutoffs for GDM diagnosis based on venous sampling can be replaced by cutoffs based on capillary sampling. A prospective cross-sectional study was performed at an antenatal care clinic including 175 pregnant women undergoing an oral glucose tolerance test (OGTT). Duplicate samples were collected by capillary and venous puncture while fasting and 1 h and 2 h after an OGTT. Both samples were analyzed on Accu-Chek Inform II. The cutoffs for a GDM diagnosis using capillary samples were corrected from 5.1 to 5.3 mmol/L for the fasting sample, from 10.0 to 11.1 mmol/L for the 1 h sample, and from 8.5 to 9.4 mmol/L for the 2-h sample using half of the dataset. Applying these cutoffs to the remaining dataset resulted in a sensitivity, specificity, and accuracy of 85.0%, 95.0%, and 90.3%, respectively, with a positive predictive value (PPV) of 83%, an negative predictive value (NPV) of 96%, and a positive negative likelihood ratio (LHR) of 16.4 using capillary sampling for the GDM diagnosis at fasting and 2-h after. Corrected cutoffs and capillary samples can be used for the diagnosis of GDM with maintained diagnostic accuracy using Accu-Chek Inform II.