Abstract Study Objectives To investigate the association between daytime napping and retinal microcirculation. Methods This is a cross-sectional study from a prospective population-based cohort. 2,662 participants were recruited after quota sampling. Information on napping was collected through face-to-face interviews. Retinal vascular calibers (RVCs), including central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE), and arterio-to-venous ratio (AVR), were obtained from fundus photography. Multivariate regression and restricted cubic spline curve were performed to determine the association between RVCs and daytime napping duration. Results 56.4% participants reported daytime napping regularly. Compared to no nap, daytime nap was related to higher CRAE, with nap duration of 0.5–1 h showing the most significant association. 0.5–1 h daytime nappers displayed an average of 4.18 µm (95% confidence interval [CI] 2.45–5.91, p < 0.001) wider CRAE than non-nappers after adjustment. No significant association was found between CRVE and daytime napping. Moreover, individuals with 0.5–1 h daytime napping had a lower risk for AVR reduction (odds ratio [OR] 0.70, 95% confidence interval [CI] 0.56–0.86, p = 0.001) than non-nappers. Similar association persisted in non-hypertensive population. Restricted cubic spline indicated a J-shaped relationship between AVR reduction and nap duration. Conclusion Retinal microcirculation was positively associated with self-reported 0.5–1 h daytime napping. Better indicators of retinal microcirculation were probably related to nap duration in a J-shaped manner. Also, the possibly beneficial role of 0.5–1 h daytime napping on retinal microcirculation might be independent of clinically diagnosed vascular diseases.