PURPOSE: To evaluate choroidal vasculature in eyes with diabetic retinopathy (DR) using a novel three-dimensional algorithm. METHODS: Patients with DR and healthy controls underwent clinical examinations and swept-source optical coherence tomography (PlexElite-9000). The choroidal layer was segmented using the ResUNet model. Phansalkar thresholding was used to binarize the choroidal vasculature. The macular area was divided into 5 sectors by a custom grid, and the 15 largest vessels in each sector were measured for mean choroidal vessel diameter (MChVD). Volumetric choroidal thickness (ChT) and the choroidal vascularity index (CVI) were calculated. A linear mixed model was used for analysis. RESULTS: This retrospective cross-sectional study analyzed 73 eyes of 45 patients with DR (36 proliferative vs. 37 nonproliferative DR, and 42 with diabetic macular edema [DME] vs. 31 without DME), and 27 eyes of 21 age-match controls. The average MChVD was decreased in DR compared with healthy (200.472 ± 28.246 µm vs. 240.264 ± 22.350 µm; P < 0.001), as well as lower sectoral MChVD (P < 0.001); however, there was no difference in average ChT between the groups (P > 0.05). The global CVI was reduced in DR, especially in temporal and central sectors (P < 0.05). Compared with nonproliferative, proliferative DR exhibited decreased ChT (temporal, P < 0.05; other sectors, P > 0.05), CVI (P > 0.05), and MChVD (P > 0.05). DME eyes demonstrated lower but not statistically significant MChVD (196.449 ± 27.221 µm vs. 205.922 ± 29.134 µm; P > 0.05) and significantly reduced average CVI (0.365 ± 0.032 vs. 0.389 ± 0.040; P = 0.008) compared with non-DME eyes. CONCLUSIONS: DR and DME eyes showed reduced MChVD and CVI, likely owing to microvascular changes leading to ischemia. These findings highlight the need for new choroidal biomarkers to better understand DR's pathogenic mechanisms.