PURPOSE. The purpose of this study was to use three-dimensional confocal microscopy to quantify the spatial patterns of capillary network alterations in nonproliferative diabetic retinopathy (NPDR). METHODS. The retinal microvasculature was perfusion-labelled in seven normal human donor eyes and six age-matched donor eyes with NPDR. The peripapillary microcirculation was studied using confocal scanning laser microscopy. Capillary density and diameters of the radial peripapillary capillary plexus (RPCP), superficial capillary plexus (SCP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP) were quantified and compared. Three-dimensional visualization strategies were also used to compare the communications between capillary beds and precapillary arterioles and postcapillary venules. RESULTS. Mean capillary diameter was significantly increased in the NPDR group (P < 0.001). Intercapillary distance was significantly increased in the DCP (P = 0.004) and RPCP (P = 0.022) of the NPDR group (P = 0.010) but not the SCP (P = 0.155) or ICP (P = 0.103). The NPDR group was associated with an increased frequency of inflow communication between the SCP and ICP/DCP and a decreased frequency of communication between the SCP and RPCP (P = 0.023). There was no difference in the patterns of outflow communications between the two groups (P = 0.771). CONCLUSIONS. This study demonstrates that capillary plexuses are nonuniformly perturbed in NPDR. These structural changes may be indicative of perturbations to blood flow patterns between different retinal layers. Our findings may aid the interpretation of previous clinical observations made using optical coherence tomography angiography as well as improve our understanding of the pathogenesis of NPDR.