Abstract Objective To analyze the relationship between dietary glycemic index (GI) and retinal microvasculature in adults. Methods This was a cross-sectional study of 300 subjects from the EVIDENT II study. Dietary GI was calculated using a validated, semi-quantitative food frequency questionnaire. Retinal photographs were digitized, temporal vessels were measured in an area 0.5–1 disc diameter from the optic disc and arteriolar-venular index (AVI) was estimated with semi-automated software. Results AVI showed a significant difference between the tertiles of GI, after adjusting for potential confounders. The lowest AVI values were observed among subjects in the highest tertile of GI, whereas the greatest were found among those in the lowest tertile (estimated marginal mean of 0.738 vs. 0.768, p = 0.014). Conclusions In adults, high dietary GI implies lowering AVI values regardless of age, gender and other confounding variables. Trial registration Clinical Trials.gov Identifier: NCT02016014 . Registered 9 December 2013.